Physical characteristics of Saccharomyces cerevisiae

We examined the physical properties of the surrounding yeast cell walls by using atomic force microscopy (AFM). The yeast cells were prepared on a cleaned glass substrate for confocal microscopy (CM) observation and were mechanically trapped into a porous membrane for AFM measurement. The confocal image of the yeast cells was measured in air, meanwhile the AFM topography images of the cells were measured in both deionized (DI) water (pH = 6.9) and phosphate‐buffered saline (PBS) solution (pH = 7.4). No significant differences between the AFM topography images of the yeast cells measured in DI water and in PBS solution could be inferred. In order to get the quantitative information on the sample elasticity, the force curves between an AFM tip and the yeast cell have been measured. These curves were measured in both DI water and in PBS solution on the same yeast cell using the same AFM cantilever to get the reliable result. The contact region of the force curve in approach mode was then converted into force versus indentation curve, which would be fitted with Hertz–Sneddon model for the calculation of the elasticity. Analysis of the curves indicates that there is a difference of the Young's modulus values of the yeast cell in various environments. These data show that the salt buffer solution increases the rigidity of the biological system. Copyright © 2008 John Wiley & Sons, Ltd.     

Shear-flow induced detachment of Saccharomyces cerevisiae from stainless steel: influence of yeast and solid surface properties

The present study focused on the shear-induced detachment of Saccharomyces cerevisiae in adhesive contact with a 316L stainless steel surface using a shear stress flow chamber, with a view to determining the respective influence of the yeast surface properties and the support characteristics. The effect of cultivation of S. cerevisiae yeast cells on their subsequent detachment from the solid surface was particularly investigated. In order to elucidate the role of stainless steel, non-metallic supports were used as control, covering a broad range of surface properties such as surface free energy and roughness: polypropylene (hydrophobic), polystyrene (mildly hydrophobic, similar to stainless steel) and glass (hydrophilic). All materials were very smooth with respect to the size of yeast. First, experiments were carried out on two types of yeast cells, just rehydrated in saline solution, a biological model widely used in the literature. The influence of the ionic strength (1.5 and 150 mM NaCl) on glass and stainless steel was evaluated. Unlike on glass, no clear evidence was found for electrostatic repulsion with stainless steel since high adhesion was observed whatever the ionic strength. A lack of correlation in adhesion results was also obtained when considering the surface physico-chemical characteristics of type I (hydrophilic) and type II (hydrophobic) rehydrated cells and those of both polymers. It was postulated that unavoidable “sticky” compounds were present on the cell wall, which could not be completely removed during the successive washings of the rehydrated cell suspension before use. This could dramatically alter the yeast surface properties and modify the adhesion strength, thus clearly demonstrating the necessity to work with yeast coming from fresh cultures. Biologically active yeast cells were then used. Once cultured, type I- and type II-yeast cells were shown to exhibit the same hydrophilic properties. Regardless of the material used, for the same ionic strength (150 mM NaCl), yeast adhesion was drastically reduced compared to rehydrated yeast cells. Among all the materials tested, the specificity of 316L stainless steel was clearly established. Indeed, for glass and polymers, cell adhesion was substratum-dependent and driven by the balance between the Lifshitz-van der Waals and Lewis acid/base interactions. Despite nearly identical surface free energies for polystyrene and stainless steel, the metallic surface promoted a totally distinct behaviour which was characterized by a strong – although highly variable – yeast adhesion.     

The interaction mechanisms between Saccharomyces cerevisiae and menadione and its application in toxicology study

This study describes interaction mechanism between menadione and the yeast with the aid of the catabolite assay and the culture fluorescence method, with a view towards exploitting the potential of the mediator in toxicology study. Double mediator system containing menadione and the hydrophilic mediator, ferricyanide, was employed for evaluating the toxic effects of furfural on the yeast and the results showed the well consistence between the MICREDOX method and the conventional method.     

Yeasts as Producers of Polyhydroxyalkanoates: Genetic Engineering of Saccharomyces cerevisiae

The genes of the poly(β-hydroxybutyrate) (PHB) synthesis pathway in Ralstonia eutropha and Methylobacterium extorquens were successfully established in the yeast Saccharomyces cerevisiae. Expression of just the polyhydroxyalkanoate (PHA) synthase gene in some experiments, and all three PHB genes (i.e., the genes encoding β-ketothiolase, acetoacetyl-CoA reductase, and PHA synthase) in others, were detected in S. cerevisiae. Thus, it can be used as a “cell factory” for the production of PHB. The maximum amount of polyester accumulated was 6.7% (wt./wt.) when all three genes were expressed. The amount of polymer accumulated in the transgenic yeast harboring just the PHA synthase gene was similar (5.2%), but slightly lower, indicating the necessity of expressing all three genes for high PHB contents in the cells. For viable production of the polymer in yeasts, more needs to be learned about the metabolism of the yeast, especially about the pathways and intermediates competing with formation of the biopolymer. Another host probably needs to be chosen.

Bacteria (on the top) with PHB inclusions and yeasts with storage compounds (on the bottom).     

Indigenous Saccharomyces cerevisiae Could Better Adapt to the Physicochemical Conditions and Natural Microbial Ecology of Prince Grape Must Compared with Commercial Saccharomyces cerevisiae FX10

Indigenous Saccharomyces cerevisiae, as a new and useful tool, can be used in fermentation to enhance the aroma characteristic qualities of the wine-production region. In this study, we used indigenous S. cerevisiae L59 and commercial S. cerevisiae FX10 to ferment Prince (a new hybrid variety from Lion Winery) wine, detected the basic physicochemical parameters and the dynamic changes of fungal communities during fermentation, and analyzed the correlations between fungal communities and volatile compounds. The results showed that the indigenous S. cerevisiae L59 could quickly adapt to the specific physicochemical conditions and microbial ecology of the grape must, showing a strong potential for winemaking. Compared with commercial S. cerevisiae FX10, the wine fermented by indigenous S. cerevisiae L59 contained more glycerol and less organic acids, contributing to a rounder taste. The results of volatile compounds indicated that the indigenous S. cerevisiae L59 had a positive effect on adding rosy, honey, pineapple and other sweet aroma characteristics to the wine. Overall, the study we performed showed that selection of indigenous S. cerevisiae from the wine-producing region as a starter for wine fermentation is conducive to improving the aroma profile of wine and preserving the aroma of the grape variety.     

SYNTHESIS AND SECRETION OF HUMAN ATRIAL NATRIURETIC PEPTIDE IN Saccharomyces cerevisiae

A chemically synthesized α-hANP gene was inserted into plasmid YFD18, which was an expression-secretion vector of yeast. The recombinant then transformed in the yeast Y33. The expression level of yeast transformants was about 700 μg ANP/L detected by RIA. More than 99% of expression products were secreted in the culture medium. N-terminal analysis of purified product showed that the first 4 amino acid residues of α-hANP were deleted.     

Genotyping of Saccharomyces cerevisiae strains by interdelta sequence typing using automated microfluidics

Amplification of genomic sequences flanked by delta elements of retrotransposons TY1 and TY2 is a reliable method for characterization of Saccharomyces cerevisiae strains. The aim of this study is to evaluate the usefulness of microfluidic electrophoresis (Caliper LabChip) to assess the factors that affect interlaboratory reproducibility of interdelta sequence typing for S. cerevisiae strain delimitation. We carried out experiments in two laboratories, using varying combinations of Taq DNA polymerases and thermal cyclers. The reproducibility of the technique is evaluated using non-parametric statistical tests and we show that the source of Taq DNA polymerase and technical differences between laboratories have the highest impact on reproducibility, whereas thermal cyclers have little impact. We also show that the comparative analysis of interdelta patterns is more reliable when fragment sizes are compared than when absolute and relative DNA concentrations of each band are considered. Interdelta analysis based on a smaller fraction of bands with intermediate sizes between 100 and 1000 bp yields the highest reproducibility.     

Production of single cell protein (SCP) from food and agricultural waste by using Saccharomyces cerevisiae

Food waste is the single-largest component of the waste stream, in order to protect and safeguard the public health, useful and innovative recycling methods are investigated. The conversion of food wastes in value-added products is becoming a more economically viable and interesting practice. Food waste, collected in the distribution sector and citrus industries, was characterised for its potential as a raw material to use in fermentation processes. In this study, the production of single-cell protein (SCP) using food waste as a substrate was investigated. The purpose of this study has been to produce SCP from mixtures of food waste using Saccharomyces cerevisiae. The main fermentation test was carried out using a 25 l bioreactor. The utilisation of food waste can allow us to not only to reduce environmental pollution, but also to obtain value-added products such as protein supply for animal feed.     

Biomethylation of tin (II) complexes in the presence of pure strains of Saccharomyces cerevisiae

The methylation of several tin compounds by strains of Saccharomyces cerevisiae strains (yeast) is described. The production of the methyl tin species was established by gas chromatography and mass spectrometry. Monomethyl tin (IV) products dominated but some dimethyl tin products were observed. This appears to be the first report of a tin methylation by a pure strain of a methylating species rather than a complex mixture obtained from an environmental source.     

Mixed Cultures of Saccharomyces kudravzevii and S. cerevisiae Modify the Fermentation Process and Improve the Aroma Profile of Semi-Sweet White Wines

The purpose of the study was to evaluate the impact of the Saccharomyces cerevisiae and S. kudriavzevii mixed culture on the fermentation, chemical and aromatic composition of semi-sweet white wines. The variables tested in the experiment were the initial ratio of yeast in mixed cultures and the time of inoculation of the S. kudriavzevii co-culture. The addition of S. kudriavzevii to the inoculum did not significantly change the chemical composition of the wines obtained. No reduction in ethanol yield was found in mixed culture fermented wines; however, in some variants of the experiment, the ethanol content was higher. The mixed cultures of S. cerevisiae and S. kudriavzevii increased the level of volatile compounds in white grape wines. Wines fermented with the co-culture of S. kudriavzevii were characterized by a more diversified ester profile. The mixed cultures of S. cerevisiae and S. kudriavzevii raised the levels of terpenes in white wines. The most promising results were obtained for mixed culture variants, in which S. kudriavzevii was sequentially inoculated on the sixth day of fermentation.     

Effects of potassium on the ethanol production rate of Saccharomyces cerevisiae carrying the plasmid pCYG4 related with ammonia assimilation

The influence of potassium on ethanol production bySaccharomyces cerevisiae wild type and AR5 cells carrying the plasmid pCYG4 was investigated. This plasmid carries the glutamate dehydrogenase gene conferring an 11-fold higher level of expressed enzyme activity over the wild type cells. All experiments were carried out in batch culture with medium supplemented to different potassium concentrations up to 180 mM. Maximum ethanol production rate was observed in the AR5 cells grown in medium supplemented with 3.5 mM of potassium ions. Glucose uptake rate increased with increasing potassium up to 60 mM, but higher concentrations depressed glucose uptake rate in both strains. Furthermore, the wild type cells showed higher growth rate, ethanol production, and glucose consumption rate than the AR5 cells. These lower rates in the AR5 cells could be explained by repression of potassium uptake by an enhancement of ammonium feeding, and greater energy requirements by these cells due the presence of the plasmid.     

光散射法快速灵敏测定和表征啤酒酵母

研究了啤酒酵母的光散射光谱. 结果表明， 在波长308.0 nm处, 光散射强度与啤酒酵母浓度在2.0×104-2.0×106 Cell/mL 范围内呈线性关系, 检出限(3σ)为4.94×102 Cell/mL. 将此方法成功地应用于培养液中啤酒酵母含量的快速、 灵敏的测定, 并对其散射光谱进行了表征.     

Development of minimal fermentation media supplementation for ethanol production using two Saccharomyces cerevisiae strains

Ethanol production by fermentation is strongly dependent on media composition. Specific nutrients, such as trace elements, vitamins and nitrogen will affect the physiological state and, consequently, the fermentation performance of the micro-organism employed. The purpose of this study has been to assess the highest ethanol production by a minimal medium, instead of the more complex nutrients supplementation used during alcoholic fermentation. All fermentation tests were carried out using a microwell plate reader to monitor the processes. Two Saccharomyces cerevisiae strains (NCYC 2826 and NCYC 3445) were tested using three nitrogen sources, supplied with different vitamin and salts. The results show that solutions made of urea phosphate, KCl, MgSO₄·7H₂O, Ca-panthothenate, biotin allowed an ethanol yield of 22.9 and 23.4 g/L for strain NCYC 2826 and NCYC 3445, respectively, representing 90 and 92% of the theoretical yield. All tests were carried out using glucose as common reference carbon source.     

Effects of low-frequency magnetic fields on the viability of yeast Saccharomyces cerevisiae

A 50 Hz magnetic field effect on the growth of yeasts Saccharomyces cerevisae was studied. The cylindrical coil induced magnetic fields with inductions up to 10 mT. Duration of exposure varied up to 24 min. Exposure took place at laboratory temperature (24-26 degrees C) and the air ventilator maintained the temperature at the place of the sample. We measured the growth curves of yeasts in broth and we calculated the number of CFU (colony forming units) on solid soil. We found that magnetic field decreases the number of yeasts, and slowed down their growth. The result is similar to the experiments with bacteria E. coli, S. aureus and L. adecarboxylata. It seems that the magnetic fields kill a part of yeasts and the bigger part of them survives and continues in their growth.     

Proteome analysis of Saccharomyces cerevisiae under metal stress by two-dimensional differential gel electrophoresis

The defense mechanism by which cells combat metal stress remains poorly understood. By utilizing a newly developed technique - the differential gel electrophoresis (DIGE) - we evaluated the biological alterations of metal stress on Saccharomyces cerevisiae at its translational level. By simultaneously comparing the differential expression profiles of thousands of proteins as results of 15 different metal treatments, we were able to closely examine the response of a large number of proteins within the yeast proteome towards individual metals, as well as the response of the same proteins towards different metals. This, to our knowledge, is the first case which demonstrates the potential of DIGE as a high-throughput tool for large-scale proteome analysis. From our studies, where yeast cells were exhaustively treated with exogenous metals, 20-30% of all proteins detected showed statistically significant changes. According to different effects (up-/downregulation) of protein expression levels observed, we were able to tentatively divide the 15 metals into three groups. By mass spectrometric analysis, more than 50 protein spots were positively identified, both quantitatively and qualitatively. One of the proteins was identified to be Cu/Zn superoxide dismutase (SOD1), and its expression levels as a result of 15 different metal treatments was further examined in greater details. Significant changes in SOD1 expression were observed throughout all 15 DIGE gels.     

Short-chain polyphosphates: Extraction effects on migration and size estimation of Saccharomyces cerevisiae extracts with polyacrylamide gel electrophoresis

Polyacrylamide gel electrophoresis is commonly used to characterize the chain length of polyphosphates (polyP), more generally called condensed phosphates. After separation, nonradioactive, optical polyP staining is limited to chain lengths greater than 15 ${\mathrm{PO}}_3^{-}$monomers with toluidine blue or 4′,6-diamidino-2-phenylindole. PolyP chain lengths longer than 62${\mathrm{PO}}_3^{-}$monomers were correlated to the shortest DNA ladders. In this study, synthetic linear polyPs (Sigma-Aldrich “Type 45”, estimated mean length of 45 ${\mathrm{PO}}_3^{-}$ monomers), trimetaphosphate (trimetaP: 3 ${\mathrm{PO}}_3^{-}$ ring), tripolyphosphate (tripolyP), pyrophosphate (PP_i), and inorganic orthophosphate (o-P_i) were visualized after separation by an in situ hydrolytic degradation process to o-P_i that was subsequently stained with methyl green. Statistically insignificant migration reduction of synthetic short-chain polyP after perchloric acid or phenol–chloroform extraction was confirmed with the Friedman test. ³¹P diffusion–ordered NMR spectroscopy confirmed that extraction also reduced PP_i diffusivity by <10%. Linear regression between the R_f peak migration value and the logarithm of synthetic polyP molecular weights enabled estimation of extracted polyP chain lengths from 2 to 45 ${\mathrm{PO}}_3^{-}$monomers. Linear polyP extracts from Saccharomyces cerevisiae grown in aerobic conditions were generally shorter than extracts cultured in anaerobic conditions. Extractions from both aerobic and anaerobic S. cerevisiae included tripolyP and o-P_i, but no PP_i.     

Metabolism of Deuterated erythro‐Dihydroxy Fatty Acids in Saccharomyces cerevisiae: Enantioselective Formation and Characterization of Hydroxylactones

Epoxides of fatty acids are hydrolyzed by epoxide hydrolases (EHs) into dihydroxy fatty acids which are of particular interest in the mammalian leukotriene pathway. In the present report, the analysis of the configuration of dihydroxy fatty acids via their respective hydroxylactones is described. In addition, the biotransformation of (±)‐erythro‐7,8‐ and ‐3,4‐dihydroxy fatty acids in the yeast Saccharomyces cerevisiae was characterized by GC/EI‐MS analysis. Biotransformation of chemically synthesized (±)‐erythro‐7,8‐dihydroxy(7,8‐²H₂)tetradecanoic acid ((±)‐erythro‐ 1 ) in the yeast S. cerevisiae resulted in the formation of 5,6‐dihydroxy(5,6‐²H₂)dodecanoic acid ( 6 ), which was lactonized into (5S,6R)‐6‐hydroxy(5,6‐²H₂)dodecano‐5‐lactone ((5S,6R)‐ 4 ) with 86% ee and into erythro‐5‐hydroxy(5,6‐²H₂)dodecano‐6‐lactone (erythro‐ 8 ). Additionally, the α‐ketols 7‐hydroxy‐8‐oxo(7‐²H₁)tetradecanoic acid ( 9a ) and 8‐hydroxy‐7‐oxo(8‐²H₁)tetradecanoic acid ( 9b ) were detected as intermediates. Further metabolism of 6 led to 3,4‐dihydroxy(3,4‐²H₂)decanoic acid ( 2 ) which was lactonized into 3‐hydroxy(3,4‐²H₂)decano‐4‐lactone ( 5 ) with (3R,4S)‐ 5 =88% ee. Chemical synthesis and incubation of (±)‐erythro‐3,4‐dihydroxy(3,4‐²H₂)decanoic acid ((±)‐erythro‐ 2 ) in yeast led to (3S,4R)‐ 5 with 10% ee. No decano‐4‐lactone was formed from the precursors 1 or 2 by yeast. The enantiomers (3S,4R)‐ and (3R,4S)‐3,4‐dihydroxy(3‐²H₁)nonanoic acid ((3S,4R)‐ and (3R,4S)‐ 3 ) were chemically synthesized and comparably degraded by yeast without formation of nonano‐4‐lactone. The major products of the transformation of (3S,4R)‐ and (3R,4S)‐ 3 were (3S,4R)‐ and (3R,4S)‐3‐hydroxy(3‐²H₁)nonano‐4‐lactones ((3S,4R)‐ and (3R,4S)‐ 7 ), respectively. The enantiomers of the hydroxylactones 4, 5 , and 7 were chemically synthesized and their GC‐elution sequence on Lipodex^® E chiral phase was determined.     

Behavior of yeast cells in aqueous suspension affected by pulsed electric field

This work discusses pulsed electric fields (PEF) induced effects in treatment of aqueous suspensions of concentrated yeast cells (S. cerevisiae). The PEF treatment was done using pulses of near-rectangular shape, electric field strength was within E=2-5 kV/cm and the total time of treatment was t(PEF)=10(-4)-0.1 s. The concentration of aqueous yeast suspensions was in the interval of C(Y)=0-22 (wt%), where 1% concentration corresponds to the cellular density of 2x10(8) cells/mL. Triton X-100 was used for studying non-ionic surfactant additive effects. The electric current peak value I was measured during each pulse application, and from these data the electrical conductivity sigma was estimated. The PEF-induced damage results in increase of sigma with t(PEF) increasing and attains its saturation level sigma approximately sigma(max) at long time of PEF treatment. The value of sigma(max) reflects the efficiency of damage. The reduced efficiency of damage at suspension volume concentration higher than phi(Y) approximately 32 vol% is explained by the percolation phenomenon in the randomly packed suspension of near-spherical cells. The higher cytoplasmic ions leakage was observed in presence of surfactant. Experiments were carried out in the static and continuous flow treatment chambers in order to reveal the effects of mixing in PEF-treatment efficiency. A noticeable aggregation of the yeast cells was observed in the static flow chamber during the PEF treatment, while aggregation was not so pronounced in the continuous flow chamber. The nature of the enhanced aggregation under the PEF treatment was revealed by the zeta-potential measurements: these data demonstrate different zeta-potential signs for alive and dead cells. The effect of the electric field strength on the PEF-induced extraction of the intracellular components of S. cerevisiae is discussed.     

Conductivity of flowing polyaniline suspensions in electric field

The formation of chain structures by polarized polyaniline (PANI) particles suspended in silicone oil in the electric field has been monitored by recording suspension conductivity in the course of time. For that purpose, three types of PANI particles differing in the conductivity (3.1 × 10⁻³, 1.7 × 10⁻¹, and 2.0 × 10⁻¹ S cm⁻¹) have been chosen out of a series of nine samples prepared by controlled protonation of PANI base in orthophosphoric acid solutions. Relaxation times reflecting this process and characterizing the rate of the response to the electric field decreased with particle conductivity, indicating a higher polarizability of particles. At the same time, the maximum conductivity of suspension increased as a consequence of the electric and shear forces acting on the particles. In the shear fields, shorter relaxation times appeared than at rest. The simultaneous measurement of the shear stress confirmed that the conductivity investigation can reliably characterize the development of electrorheological structures.     

Comparison of aroma-active volatiles and their sensory characteristics of mangosteen wines prepared by Saccharomyces cerevisiae with GC-olfactometry and principal component analysis

Mangosteen fruit is fermented with five different strains (i.e. GRE (Y1), Lalvin RC212 (Y2), Lalvin D254 (Y3), CGMCC2.23 (Y4) and CGMCC2.4 (Y5)) of the yeast Saccharomyces cerevisiae to make mangosteen wines. A total of 36 volatile compounds of the mangosteen wines were identified by gas chromatography-mass spectrometry and gas chromatography-pulsed flame photometric detection. A total of 35 odour-active compounds were identified by gas chromatography-olfactometry analysis and by the detection frequency (DF) method. The compounds with high DF values included ethyl octanoate, ethyl hexanoate and 3-methyl-2-butene-1-thiol. Principal component analysis was used to characterise the differences of the flavour profiles of those mangosteen wines. The result demonstrated that the samples could be divided into three groups that were associated closely with aroma-active compounds.