The effect of growth rate on the production and vitality of non-Saccharomyces wine yeast in aerobic fed-batch culture.

Non-Saccharomyces wine yeasts are of increasing importance due to their influence on the organoleptic properties of wine and thus the factors influencing the biomass production of these yeasts, as starter cultures, are of commercial value. Therefore, the effects of growth rates on the biomass yield (Yx/s) and fermentation performance of non-Saccharomyces yeasts at bench and pilot scale were examined. The fermentative performance and (Yx/s) were optimised, in aerobic fed-batch cultivations, to produce commercial wine seed cultures of Lachancea thermotolerans Y1240, Issatchenkia orientalis Y1161 and Metschnikowia pulcherrima Y1337. Saccharomyces cerevisiae (Lalvin EC1118) was used as a benchmark. A Crabtree positive response was shown by L. thermotolerans in a molasses-based industrial medium, at growth rates exceeding 0.21 h-1 (µcrit), resulting in a Yx/s of 0.76 g/g at 0.21 h-1 (46% of µmax) in the aerobic bioreactor-grown fed-batch culture at bench scale. At pilot scale and 0.133 h-1 (36% of µmax), this yeast exhibited ethanol concentrations reaching 10.61 g/l, as a possible result of substrate gradients. Crabtree negative responses were observed for I. orientalis and M. pulcherrima resulting in Yx/s of 0.83 g/g and 0.68 g/g, respectively, below 32% of µmax. The Yx/s of M. pulcherrima, I. orientalis and L. thermotolerans was maximised at growth rates between 0.10 and 0.12 h-1 and the fermentative capacity of these yeasts was maximised at these lower growth rates.

Adaptive response to wine selective pressures shapes the genome of a Saccharomyces interspecies hybrid.

During industrial processes, yeasts are exposed to harsh conditions, which eventually lead to adaptation of the strains. In the laboratory, it is possible to use experimental evolution to link the evolutionary biology response to these adaptation pressures for the industrial improvement of a specific yeast strain. In this work, we aimed to study the adaptation of a wine industrial yeast in stress conditions of the high ethanol concentrations present in stopped fermentations and secondary fermentations in the processes of champagne production. We used a commercial Saccharomyces cerevisiae × S. uvarum hybrid and assessed its adaptation in a modified synthetic must (M-SM) containing high ethanol, which also contained metabisulfite, a preservative that is used during wine fermentation as it converts to sulfite. After the adaptation process under these selected stressful environmental conditions, the tolerance of the adapted strain (H14A7-etoh) to sulfite and ethanol was investigated, revealing that the adapted hybrid is more resistant to sulfite compared to the original H14A7 strain, whereas ethanol tolerance improvement was slight. However, a trade-off in the adapted hybrid was found, as it had a lower capacity to ferment glucose and fructose in comparison with H14A7. Hybrid genomes are almost always unstable, and different signals of adaptation on H14A7-etoh genome were detected. Each subgenome present in the adapted strain had adapted differently. Chromosome aneuploidies were present in S. cerevisiae chromosome III and in S. uvarum chromosome VII-XVI, which had been duplicated. Moreover, S. uvarum chromosome I was not present in H14A7-etoh and a loss of heterozygosity (LOH) event arose on S. cerevisiae chromosome I. RNA-sequencing analysis showed differential gene expression between H14A7-etoh and H14A7, which can be easily correlated with the signals of adaptation that were found on the H14A7-etoh genome. Finally, we report alterations in the lipid composition of the membrane, consistent with conserved tolerance mechanisms.

Biotechnological exploitation of Saccharomyces jurei and its hybrids in craft beer fermentation uncovers new aroma combinations.

Hybridisation is an important evolutionary mechanism to bring about novel phenotypes and may produce new hybrids with advantageous combinations of traits of industrial importance. Within the Saccharomyces genus, Saccharomyces jurei is a newly discovered species and its biotechnological potential has not yet been fully explored. This yeast was found to be able to grow well in unhopped wort and at low temperatures, qualities necessary in good candidates for fermented bevarages. Here, we analysed its fermentation and aroma profile and created novel non-GMO hybrids between S. jurei and S. cerevisiae ale yeasts to develop new starter strains with interesting flavours for the craft brewing and beverage industry in general. Pilot beer fermentations with specific hybrids showed a good fermentation performance, similar to the ale parent strain, while eliminating the hyper-attenuation characteristic and a more complex flavour profile. This study exploits the genetic diversity of yeasts and shows how inter-specific hybridisation and clone selection can be effectively used in brewing to create new products and to eliminate or increase specific traits.

SnRK2.6 interacts with phytochrome B and plays a negative role in red light-induced stomatal opening.

Light is an important environmental factor for plant growth and development. Phytochrome B (phyB), a classical red/far-red light receptor, plays vital role in controlling plant photomorphogenesis and light-induced stomatal opening. Phytohormone abscisic acid (ABA) accumulates rapidly and triggers a series of physiological and molecular events during the responses to multiple abiotic stresses. Recent studies showed that phyB mutant synthesizes more ABA and exhibits improved tolerance to salt and cold stress, suggesting that a crosstalk exists between light and ABA signaling pathway. However, whether ABA signaling components mediate responses to light remains unclear. Here, we showed that SnRK2.6 (Sucrose Nonfermenting 1-Related Protein Kinase 2.6), a key regulator in ABA signaling, interacts with phyB and participates in light-induced stomatal opening. First, we checked the interaction between phyB and SnRK2s, and found that SnRK2.2/2.3/2.6 kinases physically interacted with phyB in yeast and in vitro. We also performed co-IP assay to support that SnRK2.6 interacts with phyB in plant. To investigate the role of SnRK2.6 in red light-induced stomatal opening, we obtained the snrk2.6 mutant and overexpression lines, and found that snrk2.6 mutant exhibited a significantly larger stomatal aperture under red light treatment, while the two independent overexpression lines showed significantly smaller stomatal aperture, indicative of a negative role for SnRK2.6 in red light-induced stomatal opening. The interaction of SnRK2.6 with red light receptor and the negative role of SnRK2.6 in red light-induced stomatal opening provide new evidence for the crosstalk between ABA and red light in guard cell signaling.

Frozen-dough baking potential of psychrotolerant Saccharomyces species and derived hybrids.

Despite Saccharomyces cerevisiae being a synonym for baker's yeast, the species does not perform well in all baking-related conditions. In particular, dough fermentation, or proofing, is compromised by the species' sensitivity to the low and freezing temperatures that are often used in modern bakeries. Here, screening trials that included representatives of all known Saccharomyces species, showed that S. cerevisiae was generally the most sensitive member of the genus with respect to cold and freezing conditions. We hypothesized therefore that the superior cold tolerance of the non-S. cerevisiae yeast would enable their use as frozen-dough baking strains. To test this, the different yeast species were incorporated into doughs, flash frozen and kept in a frozen state for 14 days. During the proofing stage, dough development was lower in doughs that had been frozen, relative to fresh doughs. This reduction in fermentation performance was however most pronounced with S. cerevisiae. The psychrotolerant yeasts S. eubayanus, S. jurei and S. arboricola showed a strong capacity for post-freeze proofing in terms of dough development and duration of lag phase prior to fermentation. The superior proofing power of these species resulted in breads that were significantly softer and less dense than those prepared with S. cerevisiae. A sensory panel could distinguish the S. cerevisiae and non-S. cerevisiae breads based on their physical properties, but aroma and taste were unaffected by the species employed. To further improve frozen dough baking properties, S. eubayanus, S. jurei and S. arboricola were crossed with baker's yeast through rare mating, and hybrids with improved proofing capacities in both fresh and frozen doughs relative to the parents were created. The use of S. jurei and S. arboricola in baking represents the first potential technological application of these species.

Exploring the potential of comparative de novo transcriptomics to classify Saccharomyces brewing yeasts.

In this work the potential of comparative transcriptomics was explored of Saccharomyces (S.) cerevisiae and S. pastorianus for their discrimination. This way an alternative should be demonstrated to comparative genomics, which can be difficult as a result of their aneuoploid genomes composed of mosaics of the parental genomes. Strains were selected according to their application in beer brewing, i.e. top and bottom fermenting yeasts. Comparative transcriptomics was performed for four strains each of commercially available S. cerevisiae (top fermenting) and Saccharomyces pastorianus (bottom fermenting) brewing yeasts grown at two different temperatures to mid-exponential growth phase. A non-reference based approach was chosen in the form of alignment against a de novo assembled brewery-associated pan transcriptome to exclude bias introduced by manual selection of reference genomes. The result is an analysis workflow for self-contained comparative transcriptomics of Saccharomyces yeasts including, but not limited to, the analysis of core and accessory gene expression, functional analysis and metabolic classification. The functionality of this workflow is demonstrated along the principal differentiation of accessory transcriptomes of S. cerevisiae versus S. pastorianus strains. Hence, this work provides a concept enabling studies under different brewing conditions.

Saccharomyces bayanus Enhances Volatile Profile of Apple Brandies.

Qualitative and quantitative profiles of volatiles in alcoholic beverages depend mainly on the quality of raw materials, yeasts used for fermentation, and processing technique. Saccharomyces bayanus is a yeast species which is not commonly used for the production of alcoholic beverages, but it is able to produce volatiles that add desirable aroma. Since there is little information regarding the application of that microorganism for the production of apple brandies and how it affects volatile profile of finished products, we decided to address that issue. The aim of the study was to determine the impact of S. bayanus on the profile of volatile compounds and sensory properties of apple spirits obtained from three apple cultivars (Topaz, Rubin, and Elise) in comparison to spirits obtained from fermentation carried out spontaneously or with Saccharomyces cerevisiae. Obtained brandies were analysed using gas chromatography-flame ionization detector (GC-FID), solid phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS) and sensorially. In our study, brandies produced from musts fermented by S. bayanus demonstrated the highest concentration of ethyl esters and increased concentrations of isoamyl acetate, 2-phenylethyl acetate, ethyl palmitate and hexanol. Moreover, our results support the hypothesis that non-Saccharomyces yeasts which are present during spontaneous fermentation and demonstrate higher ß-glucosidase activities enhance aroma of alcoholic beverages through releasing aroma compounds from glycosidic forms, e.g., α-phellandrene, (E)-ß-fanesene, (Z,E)-α-farnesene, α-farnesene, and farnesol. Considering results obtained in sensory analysis, we proved that S. bayanus is suitable for the production of apple brandies, improving their flavour. Brandies obtained from musts fermented by S. bayanus obtained the highest average range for "overall note" parameter in sensory analysis.

Mead Production by Saccharomyces cerevisiae Safbrew T-58 and Saccharomyces bayanus (Premier Blanc and Premier Cuvée): Effect of Cowpea (Vigna unguiculata L. Walp) Extract Concentration.

This work evaluated the effect of the cowpea (Vigna unguiculata L. Walp) extract concentration on mead production. Fermentations were carried out in 500-mL Erlenmeyer flasks containing 250 mL of honey wort (30 °Brix), supplemented with 1 g L-1 of ammonium sulfate and 0.1 g L-1 of magnesium chloride and the cowpea extract (5 and 30 g L-1), inoculated with 106 cells mL-1, and incubated at 30 °C for 240 h. Higher cell growth ((cells mL-1): 11.1 × 107, 11.3 × 107, and 19.6 × 107; substrate consumption (%): 86.0, 90.0, and 85.0) and ethanol production ((v v-1 %): 15.0, 15.5, and 14.1) for yeasts Safbrew T-58, Premier Blanc, and Premier Cuvée, respectively, were obtained with 30 g L-1 of bean extract. S. bayanus Premier Blanc had the best metabolic activity with lowest glycerol production (8.5 g L-1) and highest ethanol volumetric yields (0.51-1.52 h-1) after 48 h of fermentation.

Competition experiments in a soil microcosm reveal the impact of genetic and biotic factors on natural yeast populations.

The ability to measure microbial fitness directly in natural conditions and in interaction with other microbes is a challenge that needs to be overcome if we want to gain a better understanding of microbial fitness determinants in nature. Here we investigate the influence of the natural microbial community on the relative fitness of the North American populations SpB, SpC and SpC* of the wild yeast Saccharomyces paradoxus using DNA barcodes and a soil microcosm derived from soil associated with oak trees. We find that variation in fitness among these genetically distinct groups is influenced by the microbial community. Altering the microbial community load and diversity with an irradiation treatment significantly diminishes the magnitude of fitness differences among populations. Our findings suggest that microbial interactions could affect the evolution of yeast lineages in nature by modulating variation in fitness.

Inheritance of winemaking stress factors tolerance in Saccharomyces uvarum/S. eubayanus × S. cerevisiae artificial hybrids.

Stress has been defined as any environmental factor that impairs the growth of a living organism. High concentrations of ethanol, sugars and SO2 as well as temperature variations occurring during winemaking processes are some recognized stress factors that yeasts must overcome in order to avoid stuck or sluggish fermentations. At least two of these factors -sugar and ethanol concentrations- are strongly influenced by the global warming, which become them a worry for the future years in the winemaking industry. One of the most interesting strategies to face this complex situation is the generation of hybrids possessing, in a single yeast strain, a broader range of stress factors tolerance than their parents. In the present study, we evaluated four artificial hybrids generated with S. cerevisiae, S. uvarum and S. eubayanus using a non-GMO-generating method, in their tolerance to a set of winemaking stress factors. Their capacity to overcome specific artificial winemaking situations associated with global warming was also analyzed. All four hybrids were able to grow in a wider temperature range (8-37 °C) than their parents. Hybrids showed intermediate tolerance to higher ethanol, sugar and sulphite concentrations than their parents. Additionally, the hybrids showed an excellent fermentative behaviour in musts containing high fructose concentrations at low temperature as well as under a condition mimicking a stuck fermentation.

Joint inference and alignment of genome structures enables characterization of compartment-independent reorganization across cell types.

BACKGROUND: Comparisons of Hi-C data sets between cell types and conditions have revealed differences in topologically associated domains (TADs) and A/B compartmentalization, which are correlated with differences in gene regulation. However, previous comparisons have focused on known forms of 3D organization while potentially neglecting other functionally relevant differences. We aimed to create a method to quantify all locus-specific differences between two Hi-C data sets. RESULTS: We developed MultiMDS to jointly infer and align 3D chromosomal structures from two Hi-C data sets, thereby enabling a new way to comprehensively quantify relocalization of genomic loci between cell types. We demonstrate this approach by comparing Hi-C data across a variety of cell types. We consistently find relocalization of loci with minimal difference in A/B compartment score. For example, we identify compartment-independent relocalizations between GM12878 and K562 cells that involve loci displaying enhancer-associated histone marks in one cell type and polycomb-associated histone marks in the other. CONCLUSIONS: MultiMDS is the first tool to identify all loci that relocalize between two Hi-C data sets. Our method can identify 3D localization differences that are correlated with cell-type-specific regulatory activities and which cannot be identified using other methods.

Evolution of a novel chimeric maltotriose transporter in Saccharomyces eubayanus from parent proteins unable to perform this function.

At the molecular level, the evolution of new traits can be broadly divided between changes in gene expression and changes in protein-coding sequence. For proteins, the evolution of novel functions is generally thought to proceed through sequential point mutations or recombination of whole functional units. In Saccharomyces, the uptake of the sugar maltotriose into the cell is the primary limiting factor in its utilization, but maltotriose transporters are relatively rare, except in brewing strains. No known wild strains of Saccharomyces eubayanus, the cold-tolerant parent of hybrid lager-brewing yeasts (Saccharomyces cerevisiae x S. eubayanus), are able to consume maltotriose, which limits their ability to fully ferment malt extract. In one strain of S. eubayanus, we found a gene closely related to a known maltotriose transporter and were able to confer maltotriose consumption by overexpressing this gene or by passaging the strain on maltose. Even so, most wild strains of S. eubayanus lack native maltotriose transporters. To determine how this rare trait could evolve in naive genetic backgrounds, we performed an adaptive evolution experiment for maltotriose consumption, which yielded a single strain of S. eubayanus able to grow on maltotriose. We mapped the causative locus to a gene encoding a novel chimeric transporter that was formed by an ectopic recombination event between two genes encoding transporters that are unable to import maltotriose. In contrast to classic models of the evolution of novel protein functions, the recombination breakpoints occurred within a single functional domain. Thus, the ability of the new protein to carry maltotriose was likely acquired through epistatic interactions between independently evolved substitutions. By acquiring multiple mutations at once, the transporter rapidly gained a novel function, while bypassing potentially deleterious intermediate steps. This study provides an illuminating example of how recombination between paralogs can establish novel interactions among substitutions to create adaptive functions.

In vivo recombination of Saccharomyces eubayanus maltose-transporter genes yields a chimeric transporter that enables maltotriose fermentation.

Saccharomyces eubayanus is the non-S. cerevisiae parent of the lager-brewing hybrid S. pastorianus. In contrast to most S. cerevisiae and Frohberg-type S. pastorianus strains, S. eubayanus cannot utilize the α-tri-glucoside maltotriose, a major carbohydrate in brewer's wort. In Saccharomyces yeasts, utilization of maltotriose is encoded by the subtelomeric MAL gene family, and requires transporters for maltotriose uptake. While S. eubayanus strain CBS 12357T harbors four SeMALT genes which enable uptake of the α-di-glucoside maltose, it lacks maltotriose transporter genes. In S. cerevisiae, sequence identity indicates that maltotriose and maltose transporters likely evolved from a shared ancestral gene. To study the evolvability of maltotriose utilization in S. eubayanus CBS 12357T, maltotriose-assimilating mutants obtained after UV mutagenesis were subjected to laboratory evolution in carbon-limited chemostat cultures on maltotriose-enriched wort. An evolved strain showed improved maltose and maltotriose fermentation in 7 L fermenter experiments on industrial wort. Whole-genome sequencing revealed a novel mosaic SeMALT413 gene, resulting from repeated gene introgressions by non-reciprocal translocation of at least three SeMALT genes. The predicted tertiary structure of SeMalT413 was comparable to the original SeMalT transporters, but overexpression of SeMALT413 sufficed to enable growth on maltotriose, indicating gene neofunctionalization had occurred. The mosaic structure of SeMALT413 resembles the structure of S. pastorianus maltotriose-transporter gene SpMTY1, which has high sequences identity to alternatingly S. cerevisiae MALx1, S. paradoxus MALx1 and S. eubayanus SeMALT3. Evolution of the maltotriose transporter landscape in hybrid S. pastorianus lager-brewing strains is therefore likely to have involved mechanisms similar to those observed in the present study.

Evolutionary journey and characterisation of a novel pan-gene associated with beer strains of Saccharomyces cerevisiae.

The sequencing of over a thousand Saccharomyces cerevisiae genomes revealed a complex pangenome. Over one third of the discovered genes are not present in the S. cerevisiae core genome but instead are often restricted to a subset of yeast isolates and thus may be important for adaptation to specific environmental niches. We refer to these genes as "pan-genes," being part of the pangenome but not the core genome. Here, we describe the evolutionary journey and characterisation of a novel pan-gene, originally named hypothetical (HYPO) open-reading frame. Phylogenetic analysis reveals that HYPO has been predominantly retained in S. cerevisiae strains associated with brewing but has been repeatedly lost in most other fungal species during evolution. There is also evidence that HYPO was horizontally transferred at least once, from S. cerevisiae to Saccharomyces paradoxus. The phylogenetic analysis of HYPO exemplifies the complexity and intricacy of evolutionary trajectories of genes within the S. cerevisiae pangenome. To examine possible functions for Hypo, we overexpressed a HYPO-GFP fusion protein in both S. cerevisiae and Saccharomyces pastorianus. The protein localised to the plasma membrane where it accumulated initially in distinct foci. Time-lapse fluorescent imaging revealed that when cells are grown in wort, Hypo-gfp fluorescence spreads throughout the membrane during cell growth. The overexpression of Hypo-gfp in S. cerevisiae or S. pastorianus strains did not significantly alter cell growth in medium-containing glucose, maltose, maltotriose, or wort at different concentrations.

The impacts of Schizosaccharomyces on winemaking.

In the past century, yeasts from the genus Saccharomyces represented the only option in fermentation industries, such as winemaking, to produce wine, beer, and other fermented products. However, other genera are currently emerging to solve challenges in modern enology. Schizosaccharomyces pombe is showing promising results in solving specific challenges in northern, cool viticulture regions with highly acidic wines by deacidifying these wines through its malic acid metabolism. In addition, this microorganism is considered beneficial in warm growing regions with challenges such as the control of wine food safety problems such as the presence of biogenic amines, ochratoxin A, or ethyl carbamate. Indeed, the genus Schizosaccharomyces positively influences other important wine quality parameters, such as color and polysaccharide content. However, the main challenge of using this genus remains the selection of proper strains that alleviate problems such as the production of high acetate concentrations. Industries other than wine production such as ginger fermentation, apple wine, Kei-apple fermentation, plum wine, sparkling wine, and bilberry fermentation industries have also started to study Schizosaccharomyces species as an alternative tool for solving specific related problems. The review discusses the influence of Schizosaccharomyces on different fermentation quality parameters and its main applications in different industries.

Streptomyces Volatile Compounds Influence Exploration and Microbial Community Dynamics by Altering Iron Availability.

Bacteria and fungi produce a wide array of volatile organic compounds (VOCs), and these can act as chemical cues or as competitive tools. Recent work has shown that the VOC trimethylamine (TMA) can promote a new form of Streptomyces growth, termed "exploration." Here, we report that TMA also serves to alter nutrient availability in the area surrounding exploring cultures: TMA dramatically increases the environmental pH and, in doing so, reduces iron availability. This, in turn, compromises the growth of other soil bacteria and fungi. In response to this low-iron environment, Streptomyces venezuelae secretes a suite of differentially modified siderophores and upregulates genes associated with siderophore uptake. Further reducing iron levels by limiting siderophore uptake or growing cultures in the presence of iron chelators enhanced exploration. Exploration was also increased when S. venezuelae was grown in association with the related low-iron- and TMA-tolerant Amycolatopsis bacteria, due to competition for available iron. We are only beginning to appreciate the role of VOCs in natural communities. This work reveals a new role for VOCs in modulating iron levels in the environment and implies a critical role for VOCs in modulating the behavior of microbes and the makeup of their communities. It further adds a new dimension to our understanding of the interspecies interactions that influence Streptomyces exploration and highlights the importance of iron in exploration modulation.IMPORTANCE Microbial growth and community interactions are influenced by a multitude of factors. A new mode of Streptomyces growth-exploration-is promoted by interactions with the yeast Saccharomycescerevisiae and requires the emission of trimethylamine (TMA), a pH-raising volatile compound. We show here that TMA emission also profoundly alters the environment around exploring cultures. It specifically reduces iron availability, and this in turn adversely affects the viability of surrounding microbes. Paradoxically, Streptomyces bacteria thrive in these iron-depleted niches, both rewiring their gene expression and metabolism to facilitate iron uptake and increasing their exploration rate. Growth in close proximity to other microbes adept at iron uptake also enhances exploration. Collectively, the data from this work reveal a new role for bacterial volatile compounds in modulating nutrient availability and microbial community behavior. The results further expand the repertoire of interspecies interactions and nutrient cues that impact Streptomyces exploration and provide new mechanistic insight into this unique mode of bacterial growth.

Survival and stability of Lactobacillus fermentum and Wickerhamomyces anomalus strains upon lyophilisation with different cryoprotectant agents.

The stability of microorganisms along the time is important for allowing their industrial use as starter agents, improving fermentation processes. This study aimed to evaluate the survival and maintenance of the cell viability of the lactic acid bacteria Lactobacillus fermentum IAL 4541 and the yeast Wickerhamomyces anomalus IAL 4533, both isolated from wheat sourdough, after lyophilisation with different cryoprotectant and storage at room temperature along a year. Treatments involved adding control solution (S1 = 0.1% peptone water), and four cryoprotectant solutions S2 (10% sucrose), S3 (5% trehalose), S4 (10% skim milk powder) and S5 (10% skim milk powder plus 5% sodium glutamate) to the microbial cells previously of freeze drying processing. To verify the effect of lyophilisation on the number of microbial cells recovered, microbiological analyses were performed and cell viability was calculated before and after lyophilisation and regularly during a storage period of 365 days at room temperature. Viability after freeze-drying was influenced by the cryoprotectant agent employed, as well the microbial stability conferred along the storage. Differences on the microorganism response to some protectors were observed between the lactic acid bacteria and the yeast evaluated. W. anomalus was more affected by absence of cryoprotectant (S1) during freeze drying processing, but this microorganism was more stable than L. fermentum along the storage without the presence of protectant agents. For L. fermentum, S5 was the best protectant, allowing the recovering of 100% of the bacterial cells after lyophilisation and 87% of cell viability was observed after one year storage, followed by S4 (96 and 74%, respectively). S4 and S5 were the best protectant to W. anomalus (viability >80% after 1 year), but no increase in the yeast cell viability was conferred by addition of glutamate (S5) to skim milk. After 1 year of storage, trehalose was much more effective on protection of the yeast than bacteria (72% and 7% of viability, respectively). S2 was the less protectant agent among the tested, and their effectiveness was higher in L. fermentum (allowing 14% of cell recovering up to 120 days of storage) if compared to W. anomalus (25% of viability until 90 days of storage). Our results demonstrate that freeze-drying is a realistic technology for the stability and maintenance of the potential sourdough starter L. fermentum and W. anomalus for long time; however, the choice of cryoprotectant will influence the process effectiveness.

Fermentative behaviour and competition capacity of cryotolerant Saccharomyces species in different nitrogen conditions.

The selection of yeasts with low nitrogen requirement is a current need in winemaking. In this work, we analysed nitrogen requirements of strains belonging to the cryotolerant species S. uvarum, S. eubayanus and S. kudriavzevii, in order to evaluate their potential for conducting the fermentation of low nitrogen content grape musts. Our result demonstrated that S. eubayanus is the species less influenced by the increasing nitrogen concentrations in both growth and fermentation conditions. Strains showing the best behaviours, S. eubayanus NPCC 1285 and S. uvarum NPCC 1317, were selected to be tested in mixed cultures with S. cerevisiae T73 at different temperatures (12 °C, 20 °C and 28 °C) in synthetic grape must with different nitrogen concentrations (60, 140 and 300 mg/L YAN). The cryotolerant strains dominated the fermentations carried out at 12 °C while S. cerevisiae prevailed at 28 °C independently from the nitrogen concentration. At intermediate temperature, 20 °C, S. eubayanus mono and mixed cultures showed the best fermentative behaviour especially with low and intermediate nitrogen concentration. In summary, cryotolerant Saccharomyces species, particularly S. eubayanus, could be interesting tools to avoid fermentations stucks caused by low nitrogen content in grape musts.

Fungal bio-sorption potential of chromium in Norkrans liquid medium by shake flask technique.

In this study, the myco-reduction potential of fungi isolated from soil was ascertained by Norkrans shake flask experiment contaminated with chromium(VI). Fungal tolerance assay and induced tolerance training of the fungi were also carried out. Aspergillus niger, Penicillium, and Saccharomyces strains were isolated from the soil samples using culture based technique. Norkrans samples were collected and analyzed for Cr(VI) concentration using diphenylcarbazide spectrophotometric method. Penicillium strain was observed to be most effect at Cr(VI) concentrations of 16.1 and 8.1 mg L-1 since it was able to reduce Cr(VI) more than Saccharomyces strain and A. niger on day 20. Bio-sorption kinetics for this study was better described by pseudo second order model while Langmuir isotherm model fitted better to the equilibrium data. There was virtually steady increase in fungal growth for all the treatments through-out the experimental period. Significant negative correlation (p < 0.05) was observed between fungal growth and Cr(VI) reduction rate. The results from the induced tolerance training showed that Penicillium had the highest tolerance index (TI) values at 18, 20, and 25 mg L-1 concentrations of Cr(VI) compared to A. niger and Saccharomyces strain. These results demonstrated that these fungi have the potential to bio-absorb Cr(VI) and if properly harnessed, could be used in place of conventional remediation technology to clean-up the Cr(VI) contaminant in the field.

Amino Acid Supplementations Enhance the Stress Resistance and Fermentation Performance of Lager Yeast During High Gravity Fermentation.

The effects of different wort gravity or ethanol concentration in initial wort on the fermentation performance of lager yeast and assimilation of free amino acids (FAAs) were studied. Results showed that compared with high wort gravity (24°P), high ethanol concentration (10%, v/v) decreased yeast growth, cell viability, and wort fermentability significantly. The assimilation of FAAs was changed dramatically by high ethanol toxicity, and positive correlations between the assimilation amounts of 10 FAAs (Asp, Ser, Gly, Arg, Tyr, Val, Met, Lys, Ile, and Leu) and fermentation performance (cell viability, fermentability, and ethanol production) were identified, especially for Arg and Lys exhibiting extremely significant positive correlations. Furthermore, confirmatory testing was carried out by supplementing 24°P worts with 10 FAAs of 0.5, 1, and 2 times of their standard concentrations, respectively. Results exhibited that 10 FAA supplementations improved physiological characteristics and fermentation performance of lager yeast significantly, especially for 1 times FAA supplementation increasing wort fermentability and ethanol yield by 6 and 17%, respectively, and upregulated the expression level of HSP12 and increased more intracellular trehalose accumulation in yeast cells, indicating that stronger protective function was stimulated in yeast cells. Therefore, it was suggested that these 10 FAAs could regulate yeast cells to adapt to high gravity environmental stresses.