Dielectric Behavior of Yeast Cells Treated with HgCl2 and Cetyl Trimethyl Ammonium Bromide

The change in dielectric properties caused by the destruction of the transport barrier of yeast cells has been investigated. Dielectric measurements were made over the frequency range of 1 kc to 2 Mc by using “leaky” yeast cells prepared by treatments with HgCl₂ or CTAB (cetyl trimethyl ammonium bromide). The Hg-treated cells were observed to give smaller dielectric constants and lower critical frequencies as compared with that of the intact cells, while the CTAB-treated cells gave no clear-cut dielectric dispersion. These observations are interpreted on the basis of Maxwell-Wagner's theory as indicating the changes in the intracellular conductivity, the membrane capacitance and the membrane conductance.     

A comparative study of cell death using electrical capacitance measurements and dielectrophoresis

The changes in the dielectric properties of cells that occur during their exposure to various lethal environmental stresses were measured using both dielectric spectroscopy and dielectrophoresis. It is shown that the dielectric properties of both dying and dead yeast cells were strongly dependent on the method used to induce cell death. Methods which directly affected the membrane permeability, and consequently the membrane conductivity and internal conductivity, resulted in large changes in the suspension capacitance and dielectrophoretic behaviour, whilst methods which affected the cell interior but had little effect on the cell membrane resulted in few or no changes in the dielectric properties of the cells. The findings indicate that, depending on the method by which cell death is induced, dielectric spectroscopy may not always be able to observe differences between viable and non-viable cells, and that dielectrophoresis will not always be able to separate viable from non-viable cells.     

Electrical stimulation of the energy metabolism in yeast cells using a planar Ti-Au-Electrode interface

We report on the influence of dielectric pulse injection on the energy metabolism of yeast cells with a planar interdigitated electrode interface. The energy metabolism was measured via NADH fluorescence. The application of dielectric pulses results in a distinct decrease of the fluorescence, indicating a response of the energy metabolism of the yeast cells. The reduction of the NADH signal significantly depends on the pulse parameters, i.e., amplitude and width. Furthermore, the interface is used to detect electrical changes in the cell-electrolyte system, arising from glucose-induced oscillations in yeast cells and yeast extract, by dielectric spectroscopy at 10 kHz. These dielectric investigations revealed a β₁-dispersion for the system electrolyte/yeast cells as well as for the system electrolyte/yeast extract. In agreement with control measurements we obtained a glycolytic period of 45s for yeast cells and of 11min for yeast extract.     

Dielectric Properties of Yeast Cells Expressed With the Motor Protein Prestin

We report on the linear and nonlinear dielectric properties of budding yeast (S. cerevisiae) cells, one strain of which has been genetically modified to express prestin. This motor protein plays a crucial role in the large electromotility exhibited by the outer hair cells of mammalian inner ears. Live cell suspensions exhibit enormous dielectric responses, which can be used to probe metabolic activity, membrane potential, and other properties. The aims of this study are: (1) to compare the dielectric responses of organisms expressing prestin from those of control specimens, and (2) ultimately to further develop dielectric response as a tool to study live cells, proteins, and lipids.     

Flow-cytometric Studies of the Phagocytic Capacities of Equine Neutrophils

Methodological aspects of flow-cytometric evaluation of the phagocytic properties of equine neutrophils were elucidated. The kinetics of attachment and ingestion were studied, and the phagocytic process was more rapidly completed when serum-opsonized yeast cells were used than with use of IgG-opsonized yeast cells. Trypan blue was successfully used to quench fluorescence of non-ingested yeast cells. There were only minor differences in the kinetics of phagocytosis between quenched and un-quenched samples, indicating that attachment is rapidly followed by ingestion. Trypan blue quenching caused loss of cells with light scattering properties of granulocytes, although this did not affect the determined frequencies of truly phagocytic neutrophils. Aggregation of yeast cells proved to be a disturbance but not an obstacle to the determination of frequencies of actively phagocytic cells. Flow cytometry is well suited for studies of phagocytosis of yeast cells by equine neutrophils, and the trypan blue quenching provides a means of eliminating false-positive events due to aggregation of yeast cells. The main advantage of the flow-cytometric method is the possibility of rapid processing of a large number of samples, making the method useful for studies of herds.     

Dielectric spectroscopy of Schizosaccharomyces pombe using electrorotation and electroorientation.

Two complementary AC electrokinetic techniques electrorotation (ER) and electroorientation (EO) enabled the dielectric characterization of the rod-shaped fission yeast Schizosaccharomyces pombe. The use of microstructured electrodes allowed both ER and EO measurements to be performed over wide ranges of field frequency and medium conductivity. Due to their layered structure, living S. pombe cells exhibited up to three well resolved peaks in their ER spectra and also two distinct orientations, i.e., parallel or perpendicular to the imposed linear field. Heat treatment and enzymatic protoplast isolation led to dramatic changes in the electrokinetic behavior of fission yeast. Application of the theoretical models linking the ER and EO spectra yielded the dielectric parameters of the major structural units of S. pombe cells (cell wall, plasma membrane and cytosol). The dielectric characterization of yeasts has an enormous impact in biotechnology and biomedicine, because electric field pulse techniques (electrofusion and electropermeabilization) are widely used for production of transgenic yeast strains of economic importance. The present study also showed that combined ER and EO measurements can be employed as a powerful diagnostic tool for analyzing changes in yeast structure and physiology upon exposure to various stress conditions.     

Expression of a fungal hydrophobin in the Saccharomyces cerevisiae cell wall: effect on cell surface properties and immobilization

The aim of this work was to modify the cell surface properties of Saccharomyces cerevisiae by expression of the HFBI hydrophobin of the filamentous fungus Trichoderma reesei on the yeast cell surface. The second aim was to study the immobilization capacity of the modified cells. Fusion to the Flo1p flocculin was used to target the HFBI moiety to the cell wall. Determination of cell surface characteristics with contact angle and zeta potential measurements indicated that HFBI-producing cells are more apolar and slightly less negatively charged than the parent cells. Adsorption of the yeast cells to different commercial supports was studied. A twofold increase in the binding affinity of the hydrophobin-producing yeast to hydrophobic silicone-based materials was observed, while no improvement in the interaction with hydrophilic carriers could be seen compared to that of the parent cells. Hydrophobic interactions between the yeast cells and the support are suggested to play a major role in attachment. Also, a slight increase in the initial adsorption rate of the hydrophobin yeast was observed. Furthermore, due to the engineered cell surface, hydrophobin-producing yeast cells were efficiently separated in an aqueous two-phase system by using a nonionic polyoxyethylene detergent, C(12-18)EO(5).     

On-line monitoring of lipid storage in yeasts using impedance spectroscopy

Bioremediation technologies and many environmentally sound biosyntheses rely on the catalytic potential of whole cells. For analyzing and controlling such processes robust real-time indicators for the concentration of intact cells such as impedance are required. The conventional method measures the capacitances of cell suspensions at one or two frequencies and correlates them with biomass concentrations. However, cell inclusions such as lipid droplets or overproduced enzymes may block intracellular ion paths, thereby possibly modifying the dielectric properties of the cells. To test the hypothesis that the total impedance spectrum into the analysis may provide useful information about cell inclusions, the impedance spectrum of a technical culture of the oleaginous yeast Arxula adeninivorans was measured and evaluated every 15 s. This yeast is a good test object since it stores the excess of assimilated carbon in experimentally controllable lipid droplets. Upon correction for possible impedance signal interferences, we derived different empirical methods suitable to indicate incipient lipid formation. The methods were designed to act on-line and are thus principally suited for real-time monitoring of cell inclusions. In search for optimised bioprocess monitoring we tested a heuristic spectrum analysis using integrative statistics (RDA). With this approach we were able to accurately detect the formation of cell inclusions, which is potentially valuable for future bioprocess control strategies.     

Dielectric spectroscopy of Schizosaccharomyces pombe using electrorotation and electroorientation

Two complementary AC electrokinetic techniques electrorotation (ER) and electroorientation (EO) enabled the dielectric characterization of the rod-shaped fission yeast Schizosaccharomyces pombe. The use of microstructured electrodes allowed both ER and EO measurements to be performed over wide ranges of field frequency and medium conductivity. Due to their layered structure, living S. pombe cells exhibited up to three well resolved peaks in their ER spectra and also two distinct orientations, i.e., parallel or perpendicular to the imposed linear field. Heat treatment and enzymatic protoplast isolation led to dramatic changes in the electrokinetic behavior of fission yeast. Application of the theoretical models linking the ER and EO spectra yielded the dielectric parameters of the major structural units of S. pombe cells (cell wall, plasma membrane and cytosol). The dielectric characterization of yeasts has an enormous impact in biotechnology and biomedicine, because electric field pulse techniques (electrofusion and electropermeabilization) are widely used for production of transgenic yeast strains of economic importance. The present study also showed that combined ER and EO measurements can be employed as a powerful diagnostic tool for analyzing changes in yeast structure and physiology upon exposure to various stress conditions.     

Expression of a Fungal Hydrophobin in the Saccharomyces cerevisiae Cell Wall: Effect on Cell Surface Properties and Immobilization

The aim of this work was to modify the cell surface properties of Saccharomyces cerevisiae by expression of the HFBI hydrophobin of the filamentous fungus Trichoderma reesei on the yeast cell surface. The second aim was to study the immobilization capacity of the modified cells. Fusion to the Flo1p flocculin was used to target the HFBI moiety to the cell wall. Determination of cell surface characteristics with contact angle and zeta potential measurements indicated that HFBI-producing cells are more apolar and slightly less negatively charged than the parent cells. Adsorption of the yeast cells to different commercial supports was studied. A twofold increase in the binding affinity of the hydrophobin-producing yeast to hydrophobic silicone-based materials was observed, while no improvement in the interaction with hydrophilic carriers could be seen compared to that of the parent cells. Hydrophobic interactions between the yeast cells and the support are suggested to play a major role in attachment. Also, a slight increase in the initial adsorption rate of the hydrophobin yeast was observed. Furthermore, due to the engineered cell surface, hydrophobin-producing yeast cells were efficiently separated in an aqueous two-phase system by using a nonionic polyoxyethylene detergent, C_12-18EO₅.     

Cultivation of microorganisms in an air-solid fluidized bed fermentor with agitators

The productivity of a cell mass of Saccharomyces cerevisiae and enzymes of Eupenicillium javanicum increased by cultivation in anair-solid fluidized bed fermentor with agitators. The usefulness of the apparatus for the fluidized bed culture was verified. The productivity of amyiase and protease of the fungus by fluidized bed culture was twice as high as that by stationary culture, considering the dry weight of cells and the enzyme activity. Physiological properties of yeast cells were changed buy the fluidized bed culture; there was a decrease in the cell size of yeast and the changes to the aerobic properties of the yeast cells resulting from excessive supply of oxygen with a high flowrate of air.     

2-Oxoaldehyde metabolism in microorganisms

The properties of methylglyoxal-metabolizing enzymes in prokaryotic and eukaryotic microorganisms were studied systematically and compared with those of mammalian enzymes. The enzymes constitute a glycolytic bypass and convert methylglyoxal into pyruvate via lactate. The first step in this conversion is catalyzed by glyoxalase I, methylglyoxal reductase, or methylglyoxal dehydrogenase. The regulation of the yeast glyoxalase system was analyzed. The system was closely related to the proliferative states of yeast cells, the activity of the system being high in dividing cells and low in nondividing ones. The gene for the glyoxalase I of Pseudomonas putida and the genes responsible for the activity of glyoxalase I and methylglyoxal reductase in Saccharomyces cerevisiae were cloned and their structural and phenotypic characters studied.     

Complex Dielectric Properties of Sulfate-Reducing Bacteria Suspensions

Sulfate-reducing bacteria (SRB) can potentially enhance the remediation of heavy metals in the subsurface. Previous geophysical research has demonstrated the sensitivity of electrical measurements to SRB-mediated mineral transformation in porous media. However, the inherent dielectric properties of SRB and their direct contribution to the electrical properties of porous media are poorly understood. We studied the complex dielectric properties of SRB (Desulfovibrio vulgaris) suspensions at different concentrations and at different growth stages using a two-electrode dielectric spectroscopy measurement over the frequency range of 20 Hz to 1 MHz. Our results show higher dielectric responses (relative dielectric permittivity, real and imaginary conductivity) occurred with higher bacteria concentration at frequencies <10 kHz. Additionally, permittivity and conductivity both decreased as cells aged from mid-log phase to late stationary phase. Our results suggest that dielectric spectroscopy measurements can be used to noninvasively monitor biomass and various growth stages of SRB. Our work advances the interpretation of electrical signals associated with SRB observed in the subsurface.     

Anti-infectious potential of beta-mercapto-ethanol treated baker's yeast in gnotobiotic Artemia challenge test

Aim: To evaluate nutritional and anti‐infectious characteristics of the chemically treated baker’s yeast with 2‐mercapto‐ethanol (2ME) for gnotobiotically grown Artemia. Methods and Results: A selection of isogenic yeast strains was treated with 2ME and fed to gnotobiotically grown Artemia. In the first experiment the effect of the chemical treatment on the yeast nutritional value was studied. In most cases, 2ME‐treated yeast cells were better feed for Artemia than the untreated cells. In the second experiment, a small quantity of 2ME‐treated yeast cells was fed to Vibrio campbellii (VC) challenged Artemia. The 2ME‐treatment on some yeast strains (e.g. gas1, kre6 and chs3) significantly improved Artemia resistance against VC compared with the respective untreated yeast cells. Conclusion: Simple chemical treatment with 2ME could significantly improve the nutritional and anti‐infectious properties of some baker’s yeast strains for gnotobiotically grown Artemia. Significance and Impact of the Study: The gnotobiotic Artemia test system provides a unique opportunity (because of noninterference of other microbial compounds) to investigate how the yeast cell wall composition influences macro parameters (e.g. growth and survival) in an organism. In addition, gene expression studies in these gnotobiotically grown Artemia should provide further documentation on direct effects of yeast cells on the genes involved in immune functions.     

Non-electrostatic interactions between cultured Saccharomyces cerevisiae yeast cells and adsorbent beads in expanded bed adsorption: Influence of cell wall properties

The present work focused on the importance of performing EBA experiments under real process conditions. To this end, a high ionic strength medium was used. The influence of cultivating Saccharomyces cerevisiae yeast cells on their subsequent adsorption on a Q HyperZ anion-exchanger was investigated. Two types of industrial yeast cells were used. Once cultured, both types of cells presented similar hydrophilic surface properties and identical adsorption profiles on the anion-exchanger. This was significantly different from the results obtained in previous work on the same yeast cells, just rehydrated in saline buffer, a biological model widely used in the literature. It was postulated that unavoidable “sticky” compounds, initially present in the culture medium or formed during the drying process, were strongly adsorbed on the cell wall and 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 biomass/adsorbent interactions and the bed hydrodynamics, thus demonstrating the necessity to work with yeast coming from fresh cultures. Using biologically active yeast cells allowed to really elucidating the main physico-chemical mechanisms involved in cell adsorption by focusing on the role of the non-electrostatic interactions. Two laboratory mutant yeast strains in which the protein-related cell wall biogenesis was affected, were tested. No significant differences were observed between hydrophilic and hydrophobic yeast cells: the bed remained stable at its initial value and there was a low adsorption ratio in a narrow range between 10% and 17%. This clearly demonstrated that non-electrostatic interactions play a minor role on the affinity of yeast to anion-exchanger adsorbents.     

Der Einfluß der Flockenbildung auf die Versorgung der Hefezellen mit Sauerstoff bei der Fermentation flüssiger Kohlenwasserstoffe

Floc formation, especially the influence of floe diameter variations on the total velocity of the process, was investigated in aerobic growth processes of yeast on the hydrocarbons of crude oil. The experimental results show that the diameter of the flocs is a function of the rheological properties of the fluids and the flow conditions. The floc diameter varies between 0,1 mm and a few millimeters. About 90% of the total yeast cells are situated in the interior of the flocs. Since oxygen must be transferred to all yeast cells their oxygen supply was studied. Thus, the yeast cells in the floc interior were not sufficiently supplied with oxygen, if the floc diameter reached a critical value. In such cases a decrease of the biomass formation rate was observed, although the dissolved oxygen concentration of the aquaeous fermentation medium was greater than zero. Therefore, aerobic microbial growth processes in multicomponent systems must be carried out without floc formation or under such conditions as cause very small floc diameters.     

Glutathione production by immobilized Saccharomyces cerevisiae cells containing an ATP regeneration system

Summary Whole cells of Saccharomyces cerevisiae were immobilized in polyacrylamide gel. Consuming glucose, the immobilized cells produced glutathione from its constituent amino acids, and glutathione produced was excreted out of the gels. The conditions for immobilization of the yeast cells and for glutathione production were studied. Based on these data, the properties and the feasibility of the glycolytic pathway as an ATP regeneration system were discussed in reference to glutathione production.     

扫描介电成像技术及其在生物体系的应用

通过实例介绍了近一二年新开发的一种扫描探针显微技术——扫描介电显微镜(SDM)的基本原理、测量系统的构造和特点,并扼要地展望了它在生物体系及医学上的应用潜力.     

Molecular characterization of two ammonium transporters from the ectomycorrhizal fungus Hebeloma cylindrosporum

Heterologous expression of the yeast triple Mep mutant has enabled the first molecular characterization of AMT/MEP family members in an ectomycorrhizal fungus. External hyphae, which play a key role in nitrogen nutrition of trees, are considered as the absorbing structure of the ectomycorrhizal symbiosis and therefore molecular studies on ammonium transport in hyphae are urgently needed. The kinetic properties of AMT2 and AMT3 from Hebeloma cylindrosporum were studied in Saccharomyces cerevisiae. Expression of HcAmts in the yeast triple Mep mutant restored ammonium retention within cells. The HcAmts did not complement the ammonium sensing defect phenotype of Mep2Delta cells during pseudohyphal differentiation. Northern blot analysis in H. cylindrosporum showed that the HcAMTs were up-regulated upon nitrogen deprivation and down-regulated by ammonium.     

Physical properties of cell water in partially dried Saccharomyces cerevisiae

The equilibrium vapor pressure, the heat of vaporization, the dielectric increment, and the NMR spectra of partially dried cells were studied in Saccharomyces cerevisiae with water contents varying in the range from 25 to 0.8%. The comparative study of those physical properties suggests that physical states of the microbe can be classified into four regions in accordance with the states of the cell water: the solution region, the gel region, the mobile adsorption region, and the localized water region. Much difference in the physiological properties is found between the cells in the solution region and those in the gel region, whereas the pattern changes in physical properties take place when the cells in the gel region are dried to a further extent into the mobile or the localized region. The various modes in the molecular motion of the cell water reflected in those physical properties of the cell seem to give some insight into the biological functions of the molecule in the native as well as the dried states of the cell.