Toshiaki Arao

Effect of High-Pressure Gas on Yeast Growth

Microcalorimetry is a useful tool for monitoring the growth behavior of microorganisms. In this study, microcalorimetry was used to investigate the effects of nitrogen, air, oxygen, nitrous oxide, argon, and krypton at high pressure on the growth of the yeast Saccharomyces cerevisiae. Growth thermograms (metabolic heat vs. incubation time) were generated to estimate metabolic activity under compressed gases and to determine the 50% inhibitory pressure (IP50) and minimum inhibitory pressure (MIP), which are regarded as indices of the toxicity of compressed gases. Based on MIP values, the most toxic to the least toxic gases were found to be: O2 > N2O > air > Kr > N2 > Ar.

Effects of Compressed Hydrocarbon Gases on the Growth Activity of Saccharomyces cerevisiae

The inhibitory action of compressed hydrocarbon gases on the growth of the yeast Saccharomyces cerevisiae was investigated quantitatively by microcalorimetry. Both the 50% inhibitory pressure (IP50) and the minimum inhibitory pressure (MIP), which are regarded as indices of the toxicity of hydrocarbon gases, were determined from growth thermograms. Based on these values, the inhibitory potency of the hydrocarbon gases increased in the order methane << ethane < propane < i-butane < n-butane. The toxicity of these hydrocarbon gases correlated to their hydrophobicity, suggesting that hydrocarbon gases interact with some hydrophobic regions of the cell membrane. In support of this, we found that UV absorbing materials at 260 nm were released from yeast cells exposed to compressed hydrocarbon gases. Additionally, scanning electron microscopy indicated that morphological changes occurred in these cells.

Effects of compressed unsaturated hydrocarbon gases on yeast growth.

The effect of compressed unsaturated hydrocarbon gases on the growth of the yeast Saccharomyces cerevisiae was investigated by microcalorimetry. The growth thermograms showed that unsaturated hydrocarbon gases inhibited yeast growth. As an approach to determining the comparative toxicity of unsaturated hydrocarbon gases, we determined the 50% inhibitory pressure (IP50) and the minimum inhibitory pressure (MIP). On the basis of the IP50 and MIP values, the inhibitory potency of the gases increased in the order ethylene < propylene < 1‐butene. Additionally, scanning electron microscopy showed that cells treated with unsaturated hydrocarbon gases were damaged, including invagination of the cell surface.

Effects of saccharide in medium on stress tolerance of yeast

Effects of saccharides (glucose, fructose, galactose, mannose, lactose, maltose, sucrose, trehalose) in medium on stress tolerance of yeast (Saccharomyces cerevisiae) were studied by colony counting method. The protection effect of various saccharides for yeast cells against high pressure and high temperature was estimated with survivals. Saccharides at the concentration of 0.5 mol/l or 1.0 mol/l added to YPD culture medium apparently increased the survivals of the yeast during subjected high pressure and high temperature. Barotolerance (150 MPa, 1 hour) of the yeast cell increased with increasing concentration of saccharides. Trehalose, induced maximum barotolerance to the yeast cells and disaccharides endowed the yeast cells, with higher tolerance compared with monosaccharides. Thermotolerance (51°C, 10 min) also showed a similar pattern of survival vs. saccharide concentration, to barotolerance. The protection effects of saccharides against high pressure and high temperature were explained by the mean number of equatorial OH groups in saccharide molecule. Stress tolerance of pressure-shocked yeast in the medium containing saccharides was also estimated.

Effects of pressure on growth kinetics of protein crystals

Normal growth rates of {110} and {101} faces of tetragonal hen egg-white lysozyme crystals were measured in situ under 0.1, 50, and 100 MPa. The solubilities under high pressure enabled us to evaluate supersaturation under high pressure. We found that both growth rates decreased with pressure under the same supersaturations. This means that the surface growth kinetics depends on pressure significantly. The birth and spread model was applied to understand the pressure effects on the growth kinetics. It was found that the increase in the average ledge surface energy of the two-dimensional nuclei with pressure explained the decrease in the growth rate. The molar enthalpy of thermal denaturation of pressurized (1 hour at 100 MPa) sample (lysozyme: 5mg/ml) was also measured with a differential scanning microcalorimeter (DSC). The enthalpy was not different from that of non-pressurized sample. Thus, the effect of the irreversible pressure denaturation of lysozyme up to 100 MPa on the results of our experiments was negligible.

Effects of Gas Pressurization with Ethylene on the Ultrastructure of the Yeast Saccharomyces cerevisiae

We investigated ultrastructural changes in the yeast Saccharomyces cerevisiae when exposed to compressed ethylene gas. Transmission electron microscopy (TEM) revealed that intracellular organelles in yeast cells treated with compressed ethylene at up to 0.640 MPa (6.4 atm), especially the nuclear and plasma membranes, were seriously damaged.

Effects of compression with ethane, ethylene and their fluorinated derivatives on yeast growth

The inhibitory effect of compressed gaseous C2 compounds on yeast growth was investigated quantitatively by microcalorimetry. The growth thermograms (heat output vs. incubation time) showed that all C2 compounds tested inhibited yeast growth. After quantification of yeast growth at various pressures, we determined the 50% inhibitory pressure (IP50) and the minimum inhibitory pressure (MIP) as indices which represent the inhibitory potency of gases. The lower the IP50 and MIP values, the greater the growth inhibitory effects of the gases. Based on these values, the inhibitory potency of the gases increased in the order: ethane (C2H6) < ethylene (C2H4) < pentafluoroethane (C2HF5) < 1,1,1,2-tetrafluoroethane (C2H2F4). Furthermore, transmission electron microscopy (TEM) of yeast cells treated with compressed ethylene showed that the inner structures of the cells, especially the nuclear membrane and cytoplasmic membrane, were damaged.

Effects of High-pressure Gas on the Growth Thermograms of Yeast

Using a microcalorimeter the heat evolved during incubation of yeast cultures at 30oC was detected in the form of growth thermogram (metabolic heat — incubation time curve). Correlation of the heat evolution curves with the number of cells and the turbidity of the culture was found to be very good. In this study, the effects of high-pressure gas in open system on the growth curve of yeast were investigated and the inhibition action of the gases (nitrogen, air, nitrous oxide and argon) on the growth was quantitatively assayed using Biothermo Analyzer. The growth thermograms were used to estimate microbial activity of yeast under compressed gases and to determine minimum inhibitory pressure (MIP) that is regarded as an index of toxic potency of dissolved gases. It was found that the increase of pressure induced clear inhibitory action on yeast cells, especially for air. The order of the action was as follows: air > N2O > Ar > N2.

In situ measurements of the solubility of protein crystals under high pressure

Using a two-beam interferometer and a high-pressure cell with transparent windows, we measured the solubility of lysozyme crystals under high pressure in situ . The change in the concentration with time during equilibration was measured accurately and continuously starting from a supersaturated state (growth relaxation) and an undersaturated state (dissolution relaxation). The concentration for the dissolution relaxation reached to a constant value in a week, which did not coincide with a concentration for the growth relaxation in a comparable period. From a theoretical point of view, we regarded the asymptotic concentration for the dissolution relaxation as the solubility. The molar enthalpy of thermal denaturation of pressurized (1 hour at 100 MPa) sample (lysozyme: 5mg/ml) was also measured with a differential scanning microcalorimeter (DSC). The enthalpy was not different from that of non-pressurized sample. Thus, the effect of the irreversible pressure denaturation of lysozyme up to 100 MPa on the results of our experiments was negligible.