Growth and fermentation patterns of Saccharomyces cerevisiae under different ammonium concentrations and its implications in winemaking industry

To study the effects of assimilable nitrogen concentration on growth profile and on fermentation kinetics of Saccharomyces cerevisiae. Aims: To study the effects of assimilable nitrogen concentration on growth profile and on fermentation kinetics of Saccharomyces cerevisiae. Methods and Results: Saccharomyces cerevisiae was grown in batch in a defined medium with glucose (200 g l−1) as the only carbon and energy source, and nitrogen supplied as ammonium sulphate or phosphate forms under different concentrations. The initial nitrogen concentration in the media had no effect on specific growth rates of the yeast strain PYCC 4072. However, fermentation rate and the time required for completion of the alcoholic fermentation were strongly dependent on nitrogen availability. At the stationary phase, the addition of ammonium was effective in increasing cell population, fermentation rate and ethanol. Conclusions: The yeast strain required a minimum of 267 mg N l−1 to attain complete dryness of media, within the time considered for the experiments. Lower levels were enough to support growth, although leading to sluggish or stuck fermentation. Significance and Impact of the Study: The findings reported here contribute to elucidate the role of nitrogen on growth and fermentation performance of wine yeast. This information might be useful to the wine industry where excessive addition of nitrogen to prevent sluggish or stuck fermentation might have a negative impact on wine stability and quality

TAMMiCol: Tool for analysis of the morphology of microbial colonies.

Many microbes are studied by examining colony morphology via two-dimensional top-down images. The quantification of such images typically requires each pixel to be labelled as belonging to either the colony or background, producing a binary image. While this may be achieved manually for a single colony, this process is infeasible for large datasets containing thousands of images. The software Tool for Analysis of the Morphology of Microbial Colonies (TAMMiCol) has been developed to efficiently and automatically convert colony images to binary. TAMMiCol exploits the structure of the images to choose a thresholding tolerance and produce a binary image of the colony. The images produced are shown to compare favourably with images processed manually, while TAMMiCol is shown to outperform standard segmentation methods. Multiple images may be imported together for batch processing, while the binary data may be exported as a CSV or MATLAB MAT file for quantification, or analysed using statistics built into the software. Using the in-built statistics, it is found that images produced by TAMMiCol yield values close to those computed from binary images processed manually. Analysis of a new large dataset using TAMMiCol shows that colonies of Saccharomyces cerevisiae reach a maximum level of filamentous growth once the concentration of ammonium sulfate is reduced to 200 μM. TAMMiCol is accessed through a graphical user interface, making it easy to use for those without specialist knowledge of image processing, statistical methods or coding

Development and use of a quantum dot probe to track multiple yeast strains in mixed culture

Published 10 November 2014Saccharomyces cerevisiae strains vary in their ability to develop and enhance sensory attributes of alcoholic beverages and are often found growing in mixed strain fermentations; however, quantifying individual strains is challenging due to quantification inaccuracies, low marker longevity, and compromised kinetics. We developed a fluorescent probe, consisting of glutathione molecules conjugated to a quantum dot (QD). Two S. cerevisiae strains were incubated with different coloured probes (QD attached to glutathione molecules, QD-GSH), fermented at multiple ratios, and quantified using confocal microscopy. The QD method was compared with a culture method using microsatellite DNA analysis (MS method). Probes were taken up by an ADP1 encoded transporter, transferred from mother cell to daughter cell, detectable in strains throughout fermentation, and were non-toxic. This resulted in a new quantification method that was more accurate and efficient than the MS method.Frida S. Gustafsson, Matthew D. Whiteside, Vladimir Jiranek and Daniel M. Dural

Capturing yeast associated with grapes and spontaneous fermentations of the Negro Saurí minority variety from an experimental vineyard near León

‘Microbial terroir’ relates to the infuence of autochthonous yeasts associated with a grape cultivar on the resultant wine. Geographic region, vineyard site and topography, climate and vintage infuence the biodiversity of these microbial communities. Current research focus attempts to correlate their ‘microbial fngerprint’ to the sensorial and chemical characteristics of varietal wines from distinct geographical wine regions. This study focuses on the minor red grape variety, Negro Saurí, which has seen a resurgence in the León Appellation of Origin in Spain as a varietal wine. An experimental vineyard at Melgarajo S.A. (42° 15′ 48.68_N 5° 9′ 56.66_W) was sampled over four consecutive vintages, with autochthonous yeasts being isolated from grapes, must and pilot-scale un-inoculated fermentations, and identifed by ITS sequencing. Forty-nine isolates belonging to Metschnikowia pulcherrima, Lachancea thermotolerans, Hanseniaspora uvarum and Torulaspora delbrueckii were isolated from grapes and must, and early stages of fermentation dependent on seasonal variation. Saccharomyces cerevisiae predominated throughout fermentation, as a heterogeneous and dynamic population, with seven major biotypes identifed amongst 110 isolates across four consecutive vintages. Twenty-four S. cerevisiae isolates representing fve strains dominated in two or more vintages. Their persistence through fermentation warrants further validation of their oenological properties as starter culturesIsora González‑Alonso, Michelle Elisabeth Walker, María‑Eva Vallejo‑Pascual, Gérmán Naharro‑Carrasco and Vladimir Jirane

Diffusion-Limited Growth of Microbial Colonies.

The emergence of diffusion-limited growth (DLG) within a microbial colony on a solid substrate is studied using a combination of mathematical modelling and experiments. Using an agent-based model of the interaction between microbial cells and a diffusing nutrient, it is shown that growth directed towards a nutrient source may be used as an indicator that DLG is influencing the colony morphology. A continuous reaction-diffusion model for microbial growth is employed to identify the parameter regime in which DLG is expected to arise. Comparisons between the model and experimental data are used to argue that the bacterium Bacillus subtilis can undergo DLG, while the yeast Saccharomyces cerevisiae cannot, and thus the non-uniform growth exhibited by this yeast must be caused by the pseudohyphal growth mode rather than limited nutrient availability. Experiments testing directly for DLG features in yeast colonies are used to confirm this hypothesis

Genome-wide identification of the Fermentome; genes required for successful and timely completion of wine-like fermentation by Saccharomyces cerevisiae

BACKGROUND: Wine fermentation is a harsh ecological niche to which wine yeast are well adapted. The initial high osmotic pressure and acidity of grape juice is followed by nutrient depletion and increasing concentrations of ethanol as the fermentation progresses. Yeast's adaptation to these and many other environmental stresses, enables successful completion of high-sugar fermentations. Earlier transcriptomic and growth studies have tentatively identified genes important for high-sugar fermentation. Whilst useful, such studies did not consider extended growth (>5 days) in a temporally dynamic multi-stressor environment such as that found in many industrial fermentation processes. Here, we identify genes whose deletion has minimal or no effect on growth, but results in failure to achieve timely completion of the fermentation of a chemically defined grape juice with 200 g L-1 total sugar. RESULTS: Micro- and laboratory-scale experimental fermentations were conducted to identify 72 clones from ~5,100 homozygous diploid single-gene yeast deletants, which exhibited protracted fermentation in a high-sugar medium. Another 21 clones (related by gene function, but initially eliminated from the screen because of possible growth defects) were also included. Clustering and numerical enrichment of genes annotated to specific Gene Ontology (GO) terms highlighted the vacuole's role in ion homeostasis and pH regulation, through vacuole acidification. CONCLUSION: We have identified 93 genes whose deletion resulted in the duration of fermentation being at least 20% longer than the wild type. An extreme phenotype, 'stuck' fermentation, was also observed when DOA4, NPT1, PLC1, PTK2, SIN3, SSQ1, TPS1, TPS2 or ZAP1 were deleted. These 93 Fermentation Essential Genes (FEG) are required to complete an extended high-sugar (wine-like) fermentation. Their importance is highlighted in our Fermentation Relevant Yeast Genes (FRYG) database, generated from literature and the fermentation-relevant phenotypic characteristics of null mutants described in the Saccharomyces Genome Database. The 93-gene set is collectively referred to as the 'Fermentome'. The fact that 10 genes highlighted in this study have not previously been linked to fermentation-related stresses, supports our experimental rationale. These findings, together with investigations of the genetic diversity of industrial strains, are crucial for understanding the mechanisms behind yeast's response and adaptation to stresses imposed during high-sugar fermentations.Michelle E Walker, Trung D Nguyen, Tommaso Liccioli, Frank Schmid, Nicholas Kalatzis, Joanna F Sundstrom, Jennifer M Gardner and Vladimir Jirane

Adding functionality with additive manufacturing : fabrication of titanium-based antibiotic eluting implants

Additive manufacturing technologies have been utilised in healthcare to create patient-specific implants. This study demonstrates the potential to add new implant functionality by further exploiting the design flexibility of these technologies. Selective laser melting was used to manufacture titanium-based (Ti-6Al-4V) implants containing a reservoir. Pore channels, connecting the implant surface to the reservoir, were incorporated to facilitate antibiotic delivery. An injectable brushite, calcium phosphate cement, was formulated as a carrier vehicle for gentamicin. Incorporation of the antibiotic significantly (p=0.01) improved the compressive strength (5.8±0.7MPa) of the cement compared to non-antibiotic samples. The controlled release of gentamicin sulphate from the calcium phosphate cement injected into the implant reservoir was demonstrated in short term elution studies using ultraviolet-visible spectroscopy. Orientation of the implant pore channels were shown, using micro-computed tomography, to impact design reproducibility and the back-pressure generated during cement injection which ultimately altered porosity. The amount of antibiotic released from all implant designs over a 6hour period (<28% of the total amount) were found to exceed the minimum inhibitory concentrations of Staphylococcus aureus (16μg/mL) and Staphylococcus epidermidis (1μg/mL); two bacterial species commonly associated with periprosthetic infections. Antibacterial efficacy was confirmed against both bacterial cultures using an agar diffusion assay. Interestingly, pore channel orientation was shown to influence the directionality of inhibition zones. Promisingly, this work demonstrates the potential to additively manufacture a titanium-based antibiotic eluting implant, which is an attractive alternative to current treatment strategies of periprosthetic infections

A comparison of oral omeprazole and intravenous cimetidine in reducing complications of duodenal peptic ulcer

BACKGROUND: Gastrointestinal bleeding is a common problem and its most common etiology is peptic ulcer disease. Ulcer rebleeding is considered a perilous complication for patients. To reduce the rate of rebleeding and to fasten the improvement of patients' general conditions, most emergency departments in Iran use H2-blockers before endoscopic procedures (i.e. intravenous omeprazole is not available in Iran). The aim of this study was to compare therapeutic effects of oral omeprazole and intravenous cimetidine on reducing rebleeding rates, duration of hospitalization, and the need for blood transfusion in duodenal ulcer patients. METHODS: In this clinical trial, 80 patients with upper gastrointestinal bleeding due to duodenal peptic ulcer and endoscopic evidence of rebleeding referring to emergency departments of Imam and Sina hospitals in Tabriz, Iran were randomly assigned to two equal groups; one was treated with intravenous cimetidine 800 mg per day and the other, with 40 mg oral omeprazole per day. RESULTS: No statistically significant difference was found between cimetidine and omeprazole groups in regards to sex, age, alcohol consumption, cigarette smoking, NSAID consumption, endoscopic evidence of rebleeding, mean hemoglobin and mean BUN levels on admission, duration of hospitalization and the mean time of rebleeding. However, the need for blood transfusion was much lower in omeprazole than in cimetidine group (mean: 1.68 versus 3.58 units, respectively; p < 0.003). Moreover, rebleeding rate was significantly lower in omeprazole group (15%) than in cimetidine group (50%) (p < 0.001). CONCLUSION: This study demonstrated that oral omeprazole significantly excels intravenous cimetidine in reducing the need for blood transfusion and lowering rebleeding rates in patients with upper gastrointestinal bleeding. Though not statistically significant (p = 0.074), shorter periods of hospitalization were found for omeprazole group which merits consideration for cost minimization

Computational Models for Prediction of Yeast Strain Potential for Winemaking from Phenotypic Profiles

Saccharomyces cerevisiae strains from diverse natural habitats harbour a vast amount of phenotypic diversity, driven by interactions between yeast and the respective environment. In grape juice fermentations, strains are exposed to a wide array of biotic and abiotic stressors, which may lead to strain selection and generate naturally arising strain diversity. Certain phenotypes are of particular interest for the winemaking industry and could be identified by screening of large number of different strains. The objective of the present work was to use data mining approaches to identify those phenotypic tests that are most useful to predict a strain's potential for winemaking. We have constituted a S. cerevisiae collection comprising 172 strains of worldwide geographical origins or technological applications. Their phenotype was screened by considering 30 physiological traits that are important from an oenological point of view. Growth in the presence of potassium bisulphite, growth at 40 degrees C, and resistance to ethanol were mostly contributing to strain variability, as shown by the principal component analysis. In the hierarchical clustering of phenotypic profiles the strains isolated from the same wines and vineyards were scattered throughout all clusters, whereas commercial winemaking strains tended to co-cluster. Mann-Whitney test revealed significant associations between phenotypic results and strain's technological application or origin. Naive Bayesian classifier identified 3 of the 30 phenotypic tests of growth in iprodion (0.05 mg/mL), cycloheximide (0.1 mu g/mL) and potassium bisulphite (150 mg/mL) that provided most information for the assignment of a strain to the group of commercial strains. The probability of a strain to be assigned to this group was 27% using the entire phenotypic profile and increased to 95%, when only results from the three tests were considered. Results show the usefulness of computational approaches to simplify strain selection procedures.Ines Mendes and Ricardo Franco-Duarte are recipients of a fellowship from the Portuguese Science Foundation, FCT (SFRH/BD/74798/2010, SFRH/BD/48591/2008, respectively) and Joao Drumonde-Neves is recipient of a fellowship from the Azores government (M3.1.2/F/006/2008 (DRCT)). Financial support was obtained from FEDER funds through the program COMPETE and by national funds through FCT by the projects FCOMP-01-0124-008775 (PTDC/AGR-ALI/103392/2008) and PTDC/AGR-ALI/121062/2010. Lan Umek and Blaz Zupan acknowledge financial support from Slovene Research Agency (P2-0209). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.info:eu-repo/semantics/publishedVersio