John Kapolos

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 degrees C, 25 degrees C, 20 degrees C, and 15 degrees 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 degrees 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.

THE STUDY OF THE EFFECT OF FERMENTATION TEMPERATURE ON THE GROWTH KINETICS OF SACCHAROMYCES CEREVISIAE YEAST STRAIN, IN THE PRESENCE OR ABSENCE OF SUPPORT, BY CHROMATOGRAPHIC TECHNIQUES

The Gravitational field-flow fractionation (GFFF) in combination with the conventional gas chromatography and high-performance liquid chromatography were applied in order to study the effect of temperature (15°, 20°, 25°, and 30°C) on the fermentation kinetics and the proliferation of the alcohol-resistant and psychrophilic Saccharomyces cerevisiae (AXAZ-1) yeast strain, in the presence or absence of wheat starch granules as immobilization carrier. The results indicated that both systems (free and immobilized cells) performed better at 30°C, whereas at lower temperatures decreases in the fermentation rate and in the number of viable cells were observed. The duration and the rate of the fermentation process were shorter in the case of immobilized cells as compared with free cells, which is in accordance with literature. Moreover, wheat starch granules proved to be capable and efficient for the immobilization process as it is a cheap material, of food grate purity, available in large amounts. The activation energy for the fermentation process (Ea) was reduced in the case of immobilized cells as compared with free cells.

THE STUDY OF THE INFLUENCE OF TEMPERATURE AND INITIAL GLUCOSE CONCENTRATION ON THE FERMENTATION PROCESS IN THE PRESENCE OF Saccharomyces cerevisiae YEAST STRAIN IMMOBILIZED ON STARCH GELS BY REVERSED-FLOW GAS CHROMATOGRAPHY

The technique of reversed-flow gas chromatography (RFGC) was employed for the determination of the alcoholic fermentation phases and of kinetic parameters for free and immobilized cell systems, at different initial glucose concentrations and temperature values. In addition to this, due to its considerable advantages over other techniques, RFGC was used for the characterization of a new biocatalyst, yeast cells immobilized on starch gel, and especially wheat starch gel. Immobilization of wine yeast Saccharomyces cerevisiae AXAZ-1 was accomplished on wheat and corn starch gels in order to prepare new biocatalysts with great interest for the fermentation industry. The RFGC led with great accuracy, resulting from a literature review, to the determination of reaction rate constants and activation energies at each phase of the fermentation processes. A maximum value of rate constants was observed at initial glucose concentration of 205 g/L, where a higher number of yeast cells was observed. The increase of glucose concentrations had a negative influence on the growth of AXAZ-1 cells and rate constants were decreased. The decrease of fermentation temperature caused a substantial reduction in the viability of immobilized cells as well as in rate constant values. Activation energies of corn starch gel presented lower values than those of wheat starch gel. However, the two supports showed higher catalytic efficiency than free cell systems, proving that starch gels may act as a promoter of the catalytic activity of the yeast cells involved in the fermentation process.

Kinetic study of aggregation of milk protein and/or surfactant-stabilized oil-in-water emulsions by sedimentation field-flow fractionation.

Milk proteins are able to facilitate the formation and stabilization of oil droplets in food emulsions. This study employed Sedimentation Field-Flow Fractionation (SdFFF) to monitor changes in particle size distribution of freshly prepared emulsions with varying weight contributions of sodium caseinate (SC) and whey protein concentrate (WPC). The effect of the addition of Tween 80 (T) on the initial droplet size was also investigated. The results indicated that emulsifying ability follows the order Tween 80>WPC>SC, with corresponding weight average droplet diameter of 0.319, 0.487 and 0.531μm respectively, when each of the above emulsifiers was used solely. The stability of sodium caseinate emulsions was studied at 30.5 and 80.0°C by measuring the particle size distribution for a period of 70h. Emulsions withstood the temperatures and exhibited an initial increase in particle size distribution caused by heat-induced droplet aggregation, followed by a decrease to approximately the initial droplet size. The rate of droplet aggregation depends on the severity of thermal processing, as revealed by the kinetics of particle aggregation during aging at different temperatures. Comparison of the experimental rate constants found from SdFFF, with those determined theoretically gives invaluable information about the oil droplet stability and the aggregation mechanism. Based on the proposed mechanistic scheme various physicochemical quantities, which are very important in explaining the stability of oil-in-water emulsions, were determined. Finally, the advantages of SdFFF in studying the aggregation of the oil-in-water droplets, in comparison with other methods used for the same purpose, are discussed.

Characterization of Polymeric Coatings in Terms of Their Ability to Protect Marbles and Clays against Corrosion from Sulfur Dioxide by Inverse Gas Chromatography

The protective performance of polymeric coatings was tested on marble and clays against their corrosion from sulfur dioxide. The method used was reversed-flow gas chromatography, which allowed us to determine deposition velocities (V d ), total effective diffusion coefficients (D y ), and local adsorbed equilibrium concentrations of SO2 on marbles and clays both in the presence and in the absence of the coating. The protective efficiency of a model polymeric coating, namely the acrylic copolymer Paraloid B-72, on marbles and clays against corrosion from SO2 was estimated in terms of the parameters Vd, Dy, and .

SEDIMENTATION FIELD-FLOW FRACTIONATION AS A TOOL FOR THE STUDY OF MILK PROTEIN-STABILIZED MODEL OIL-IN-WATER EMULSIONS: EFFECT OF PROTEIN CONCENTRATION AND HOMOGENIZATION PRESSURE

Milk protein-stabilized model emulsions were formed using high-pressure homogenization and the effect of protein concentration and homogenization pressure during emulsification on the particle size was studied. Various techniques are available for determining particle size distribution, each one of which has its own advantages and disadvantages. In this study, sedimentation field-flow fractionation was employed for the size characterization of oil-in water emulsion droplets. Increasing protein content results in significant reduction in emulsion particle size for the concentration range (0.5–3.0% w/w) employed in this study. Low protein content (<1%) may be correlated with bridging flocculation leading to increased particle size, as indicated by optical microscopy. Similarly, increasing pressure during the homogenization process results in decreasing significantly the particle size of the oil-in-water emulsions, for the pressure range (20–60 MPa) utilized in this study. Increased heating associated with high levels of pressure during the homogenization process, can result in changes in the oil or protein structure, which in turn may have an impact on the physicochemical properties of the oil-in-water emulsions on a long-term basis. The results of this study indicate that sedimentation field-flow fractionation can be employed to reliably monitor changes in particle size of model emulsions induced by protein concentration and homogenization pressure. The technique can be a valuable tool for understanding the properties of colloidal systems and therefore its implementation in emulsion/dispersion technology laboratories is certainly justified.

Influence of pH and Initial Glucose Concentration on the Growth of Saccharomyces cerevisiae Yeast Strain by Gravitational Field Flow Fractionation

Gravitational field-flow fractionation (GrFFF) was applied for the study of the growth of Saccharomyces cerevisiae yeast strain, in the presence or the absence of corn and wheat starch granules as immobilization carriers. Fermentations were conducted at different values of pH (3.0, 4.0, 5.0, and 6.0) and initial glucose concentration (177 g/L, 205 g/L, 247 g/L and 300 g/L) in order to find the most favorable situation for the growth and proliferation of Saccharomyces cerevisiae cells. The distinction of the phases of the yeast cell cycle was also succeeded by the same technique. The results indicate that the growth of yeast cells was enhanced at pH 5.0 and glucose concentrations of 177 g/L and 205 g/L. Higher glucose concentrations (247 g/L and 300 g/L) acted as an inhibitor to cell proliferation. Immobilization on wheat starch provided wider peak profiles, suggesting a broad size of cells and lower concentrations of haploid cells comparing to cells immobilized on corn starch granules. The determination of Michaelis-Menten constants for free and immobilized cells proved the higher affinity of immobilized cells towards the substrate. The values found were in good agreement with those reported in literature.

Reversed – Flow Gas Chromatography as a tool for studying the interaction between aroma compounds and starch

The versatile technique of reversed-flow gas chromatography was introduced to calculate physicochemical quantities for the interaction between aroma compounds and starch. Adsorption, adsorption/desorption, and surface reaction rate constants as well as surface diffusion coefficients for the vapors of aroma compounds over the different starch surfaces were calculated in the temperature range of 303.15-333.15 K. Enthalpies of adsorption between -45.5 and -109.0 kJ mol-1 and enthalpies of physicochemical interaction between 6.8 and 47.4 kJ mol-1 were also calculated for all the systems studied. From the obtained results, it is concluded that the interaction forces between aroma compounds and starch correspond to weak energy bonds such as hydrogen bonds and dipole-dipole interactions. For all the systems studied, except for the system heptanal/potato, physical sorption of aroma compounds on starch granules was indicated according to the calculated activation energies.

Study of the influence of surfactants on the activity coefficients and mass transfer coefficients of methanol in aqueous mixtures by reversed-flow gas chromatography

This work focuses on the influences of surfactants on the activity coefficients, γ, of methanol in binary mixtures with water, as well as on the mass transfer coefficients, kc, for the evaporation of methanol, which is a ubiquitous component in the troposphere, from mixtures of methanol with water at various surfactants and methanols concentrations. The technique used is the Reversed-Flow Gas Chromatography (R.F.G.C.), a version of Inverse Gas Chromatography, which allows determining both parameters by performing only one experiment for the kc parameter and two experiments for the γ parameter. The kc and γ values decrease in the presence of the three surfactants used (CTAB, SDS, TRITON X-100) at all methanols and surfactants concentrations. The decrease in the methanols molar fraction, at constant number of surfactant films leads to a decrease in the kc and γ values, while the decrease in the surfactants concentration, at constant methanols molar fraction leads to an increase in both the kc and γ parameters. Mass transfer coefficients for the evaporation of methanol at the surfactant films, are also calculated which are approximately between 4 and 5 orders of magnitude larger than the corresponding mass transfer coefficients at the liquid films. Finally, thicknesses of the boundary layer of methanol in the mixtures of methanol with water were determined. The quantities found are compared with those given in the literature or calculated theoretically using various empirical equations. The precision of the R.F.G.C. method for measuring γ and kc parameters is approximately high (94.3-98.0%), showing that R.F.G.C. can be used with success not only for the thermodynamic study of solutions, but also for the interphase transport.