The influence of inert solids on ethanol production by Saccharomyces cerevisiae

The catalytic role of various inert solid supports on acceleration of alcohol fermentation by Saccharomyces cerevisiae was investigated. The enhanced rate of alcohol production was dependent on the nature of the support as well as on the amount used. Among all the tested supports, chitosan flakes showed the maximum yield of alcohol (93% of theoretical yield). This higher rate of alcohol production was associated with the twofold increase in the activity of alcohol dehydrogenase over control. Our results suggest that the addition of a small fraction of solids in submerged fermentations to facilitate cell anchorage for enhanced metabolic activity is easier and more economical compared to cell immobilization processes. IICT Communication No. 4266. Some of the results in this article are covered under a patent.     

New separation methodologies for the distinction of the growth phases of Saccharomyces cerevisiae cell cycle

In the present work two separation techniques, namely the gravitational field-flow fractionation (GrFFF) and the reversed-flow gas chromatography (RFGC), are proposed for the distinction of the growth phases of Saccharomyces cerevisiae (AXAZ-1) yeast cycle at different temperatures (30 °C, 25 °C, 20 °C, and 15 °C) and pH (2.0, 3.0, 4.0 and 5.0) values. During the fermentation processes, differences observed in the peak profiles, obtained by GrFFF, can be related with the unlike cell growth. The distinction of the phases of AXAZ-1 cell cycle with the GrFFF, was also confirmed with the RFGC technique, which presented similar fermentation time periods for the alcoholic fermentation phases. Simultaneously, the reaction rate constant for each phase of the fermentation process and the activation energies were determined with the aid of the RFGC technique. Finally, the application of both the GrFFF and the RFGC techniques, in combination with high-performance liquid chromatography, allowed us to find the ideal experimental conditions (temperature and pH) for the alcoholic fermentation by AXAZ-1. The results indicate that S. cerevisiae cells performed better at 30 °C, whereas at lower temperatures decreases in the fermentation rate and in the number of viable cells were observed. Moreover, the pH of the medium (pH 5.0) resulted in higher fermentation rates and ethanol productivities.     

Elastic properties of chemically modified baker's yeast cells studied by AFM

Chemical pretreatment is widely used to facilitate transformation of living cells when foreign components are introduced into a cell through the cell wall. The influence of appropriate chemicals on the wall properties and mechanism of transformation is still a matter of intensive studies. Saccharomyces cerevisiae cells (also known as baker's yeast) were investigated by atomic force microscopy (AFM). The cell walls were modified by lithium acetate and dithiothreitol. The AFM imaging was performed in liquid water‐based environment. The living cells were fixed by trapping into the holes of a polycarbonate membrane. Mechanical and morphological properties of initial intact cells and treated cells were investigated. The increased stiffness of the chemically treated cells was observed. As deduced from the applied theoretical Hertz‐Sneddon model, the treated cells show completely different response mechanism to applied mechanical pressure in comparison with the intact cells. Also, the increased roughness of the cell wall of the treated yeasts was observed. Copyright © 2011 John Wiley & Sons, Ltd.     

Spectrophotometric flow injection monitoring of sulfide during sugar fermentation

A spectrophotometric flow injection procedure involving N,N-dimethyl-p-phenylenediamine (DMPD) is applied to the sulfide monitoring of a sugar fermentation by Saccharomyces cerevisiae under laboratory conditions. The gaseous chemical species evolving from the fermentative process, mainly CO₂, are trapped allowing a cleaned sample aliquot to be collected and introduced into the flow injection analyzer. Measurement rate, signal repeatability, detection limit and reagent consumption per measurement were estimated as 150 h⁻¹, 0.36% (n = 20), 0.014 mg L⁻¹ S and 120 μg DMPD, respectively. The main characteristics of the monitoring record are discussed. The strategy is worthwhile for selecting yeast strain, increasing the industrial ethanol production and improving the quality of wines.     

Stereo‐Inversion in the (4R)‐γ‐Decanolactone Synthesis by Saccharomyces cerevisiae: (2E,4S)‐4‐Hydroxydec‐2‐enoic Acid Acts as a Key Intermediate

Methyl (2E,4R)‐4‐hydroxydec‐2‐enoate, methyl (2E,4S)‐4‐hydroxydec‐2‐enoate, and ethyl (±)‐(2E)‐4‐hydroxy[4‐²H]dec‐2‐enoate were chemically synthesized and incubated in the yeast Saccharomyces cerevisiae. Initial C‐chain elongation of these substrates to C₁₂ and, to a lesser extent, C₁₄ fatty acids was observed, followed by γ‐decanolactone formation. Metabolic conversion of methyl (2E,4R)‐4‐hydroxydec‐2‐enoate and methyl (2E,4S)‐4‐hydroxydec‐2‐enoate both led to (4R)‐γ‐decanolactone with >99% ee and 80% ee, respectively. Biotransformation of ethyl (±)‐(2E)‐4‐hydroxy(4‐²H)dec‐2‐enoate yielded (4R)‐γ‐[²H]decanolactone with 61% of the ²H label maintained and in 90% ee indicating a stereoinversion pathway. Electron‐impact mass spectrometry analysis (Fig. 4) of 4‐hydroxydecanoic acid indicated a partial C(4)→C(2) ²H shift. The formation of erythro‐3,4‐dihydroxydecanoic acid and erythro‐3‐hydroxy‐γ‐decanolactone from methyl (2E,4S)‐4‐hydroxydec‐2‐enoate supports a net inversion to (4R)‐γ‐decanolactone via 4‐oxodecanoic acid. As postulated in a previous work, (2E,4S)‐4‐hydroxydec‐2‐enoic acid was shown to be a key intermediate during (4R)‐γ‐decanolactone formation via degradation of (3S,4S)‐dihydroxy fatty acids and precursors by Saccharomyces cerevisiae.     

Fluorescence staining of yeast cells permeabilized by killer toxin K1: Determination of optimum conditions

Optimal assay conditions were established for the previously described method used to determine the activity ofSaccharomyces cerevisiae pore-forming killer toxin K1. The method is based on cell staining with bromocresol purple. Sensitive cells ofS. cerevisiae from the early exponential phase under nongrowth conditions and in the presence of glucose were the most convenient for determining the killer toxin activity. Maximum killing war reached when the suspension was buffered with 10 mM citrate-phosphate at pH 4.6.     

Nuclear Magnetic Resonance Metabolomics with Double Pulsed-Field-Gradient Echo and Automatized Solvent Suppression Spectroscopy for Multivariate Data Matrix Applied in Novel Wine and Juice Discriminant Analysis

The quality of foods has led researchers to use various analytical methods to determine the amounts of principal food constituents; some of them are the NMR techniques with a multivariate statistical analysis (NMR-MSA). The present work introduces a set of NMR-MSA novelties. First, the use of a double pulsed-field-gradient echo (DPFGE) experiment with a refocusing band-selective uniform response pure-phase selective pulse for the selective excitation of a 5–10-ppm range of wine samples reveals novel broad ¹H resonances. Second, an NMR-MSA foodomics approach to discriminate between wine samples produced from the same Cabernet Sauvignon variety fermented with different yeast strains proposed for large-scale alcohol reductions. Third a comparative study between a nonsupervised Principal Component Analysis (PCA), supervised standard partial (PLS-DA), and sparse (sPLS-DA) least squares discriminant analysis, as well as orthogonal projections to a latent structures discriminant analysis (OPLS-DA), for obtaining holistic fingerprints. The MSA discriminated between different Cabernet Sauvignon fermentation schemes and juice varieties (apple, apricot, and orange) or juice authentications (puree, nectar, concentrated, and commercial juice fruit drinks). The new pulse sequence DPFGE demonstrated an enhanced sensitivity in the aromatic zone of wine samples, allowing a better application of different unsupervised and supervised multivariate statistical analysis approaches.     

Long range correlation and possible electron conduction through DNA sequences

Long range correlation analysis and charge conductivity investigation are applied to sequences in 16 chromosomes in the Saccharomyces cerevisiae genome. DNA sequence data are analyzed via Hurst’s analysis and Detrended Fluctuation Analysis (DFA) analysis. Super diffusive nature of mapping sequences are evident with the measured Hurst exponent H to be around the value of 0.60 for all sequences in the 16 chromosomes. The DFA result is consistent with the result from the Hurst analysis. Tight binding models are applied for the investigation of charge conduction through DNA sequences. The overall averaged transmission coefficients, 〈T_N〉_av, calculated from sixteen chromosomes are shown to be significantly different from values calculated from random as well as periodic sequences. Sequences from the S. cerevisiae genome promise better charge conduction ability than random sequences. Finally, delocalized electronic wave function patterns are also shown through calculations using the tight binging model. Slightly delocalized electronic wavefunctions are seen on sequences in sixteen chromosomes, as compared with those obtained from random sequences on the same eigenenergies.     

Analysis of steroids in yeast-mediated cell culture by on-line solid-phase extraction coupled high-performance liquid chromatography electrospray-ionization/mass spectrometry and novel continuous postcolumn infusion of internal standard technique

The reduction of 17-ketosteroid estrone or androstenedione to corresponding 17α- and 17β-estradiol or testosterone and epitestosterone has been performed with Saccharomyces cerevisiae. In the analysis of the cell culture, the solid-phase extraction (SPE) method was on-line coupled to high-performance liquid chromatography electrospray-ionization/mass spectrometry (HPLC-ESI/MS) for sample pretreatment to eliminate the complicated matrix interference and preconcentrate of the analytes before chromatographic separation. A novel quantification method with the continuous postcolumn infusion of internal standard was developed for the determination of substrate and products. This novel quantitative method can stabilize and enhance the ionization of all analytes during analysis. The HPLC-ESI/MS analysis of estrone, 17α-, and 17β-estradiol was operated with a negative ion mode and the analysis of androstenedione, testosterone, and epitestosterone was operated with a positive ion mode. The optimal concentration of the internal standard progesterone with the continuous postcolumn infusion technique was 3 μg mL⁻¹ for estrogen analysis and 1 ng mL⁻¹ for androgen analysis and both were at a constant infusion rate of 0.5 μL min⁻¹. All of the linear correlation coefficients of the standard calibration curves were over 0.99 and had a linear range from 0 to 50 ng mL⁻¹. The limit of detections (LODs) and the limit of quantitations (LOQs) for steroids analyzed were from 0.12 to 0.36 ng mL⁻¹ and from 0.4 to 1.2 ng mL⁻¹, respectively. The analysis accuracies and precisions were better than 94% and lower than 8.8% R.S.D., respectively. The developed method for the analysis of steroids in the cell culture was successful.     

A chemiluminescence biochemical oxygen demand measuring method

A new chemiluminescence biochemical oxygen demand (BOD_CL) determining method was studied by employing redox reaction between quinone and Baker's yeast. The measurement was carried out by utilizing luminol chemiluminescence (CL) reaction catalyzed by ferricyanide with oxidized quinone of menadione, and Saccharomyces cerevisiae using a batch-type luminometer. In this study, dimethyl sulfoxide was used as a solvent for menadione. After optimization of the measuring conditions, the CL response to hydrogen peroxide in the incubation mixture had a linear response between 0.1 and 100 μM H₂O₂ (r² = 0.9999, 8 points, n = 3, average of relative standard deviation; R.S.D._av = 4.22%). Next, a practical relationship between the BOD_CL response and the glucose glutamic acid concentration was obtained over a range of 11-220 mg O₂ L⁻¹ (6 points, n = 3, R.S.D._av 3.71%) with a detection limit of 5.5 mg O₂ L⁻¹ when using a reaction mixture and incubating for only 5 min. Subsequently, the characterization of this method was studied. First, the BOD_CL responses to 16 pure organic substances were examined. Second, the influences of chloride ions, artificial seawater, and heavy metal ions on the BOD_CL response were investigated. Real sample measurements using river water were performed. Finally, BOD_CL responses were obtained for at least 8 days when the S. cerevisiae suspension was stored at 4 °C (response reduction, 69.9%; R.S.D. for 5 testing days, 18.7%). BOD_CL responses after 8 days and 24 days were decreased to 69.9% and 35.8%, respectively, from their original values (R.S.D. for 8 days involving 5 testing days, 18.7%).