Improvement of alcoholic fermentation by calcium ions under enological conditions involves the increment of plasma membrane H<Superscript>+</Superscript>-ATPase activity

The effect of Ca²⁺ on alcoholic fermentation and plasma membrane H⁺-ATPase activity of wine yeast under enological conditions were investigated in this study. The results showed that fermentation rate, cell growth and ethanol production were improved by 0.5 and 1.5 mM Ca²⁺ supplementation, which correlated well with the increment of ATPase activity and protein levels. Considering the important role of ATPase in the tolerance of yeast to ethanol, the improvement could be, at least partially, attributed to the increment of ATPase activity. No activation of ATPase by Ca²⁺ was observed in the early phase of fermentation and the increment of activity was only observed when ethanol concentration exceeded 6.5%. Therefore, the enhancement of ATPase activity by Ca²⁺ was ascribed to alleviating the inhibition of ATPase activity by ethanol through protection of membrane structure. Our results suggest that, besides maintenance of cell membrane structure, the increment of plasma membrane ATPase activity was also responsible for the improvement of alcoholic fermentation by Ca²⁺ supplementation.     

Ethanol tolerance in bacteria

The adverse effects of ethanol on bacterial growth, viability, and metabolism are caused primarily by ethanol-induced leakage of the plasma membrane. This increase in membrane leakage is consistent with known biophysical properties of membranes and ethanolic solutions. The primary actions of ethanol result from colligative effects of the high molar concentrations rather than from specific interactions with receptors. The ethanol tolerance of growth in different microorganisms appears to result in large part from adaptive and evolutionary changes in cell membrane composition. Different cellular activities vary in their tolerance to ethanol. Therefore, it is essential that the aspect of cellular function under study be specifically defined and that comparisons of ethanol tolerance among systems share this common definition. Growth is typically one of the most sensitive cellular activities to inhibition by ethanol, followed by survival, or loss of reproductive ability. Glycolysis is the most resistant of these three activities. Since glycolysis is an exergonic process, a cell need not be able to grow or remain viable for glycolysis to occur.     

In vivo activation by ethanol of plasma membrane ATPase of Saccharomyces cerevisiae.

Ethanol, in concentrations that affect growth and fermentation rates (3 to 10% [vol/vol]), activated in vivo the plasma membrane ATPase of Saccharomyces cerevisiae. The maximal value for this activated enzyme in cells grown with 6 to 8% (vol/vol) ethanol was three times higher than the basal level (in cells grown in the absence of ethanol). The Km values for ATP, the pH profiles, and the sensitivities to orthovanadate of the activated and the basal plasma membrane ATPases were virtually identical. A near-equivalent activation was also observed when cells grown in the absence of ethanol were incubated for 15 min in the growth medium with ethanol. The activated state was preserved after the extraction from the cells of the membrane fraction, and cycloheximide appeared to prevent this in vivo activation. After ethanol removal, the rapid in vivo reversion of ATPase activation was observed. While inducing the in vivo activation of plasma membrane ATPase, concentrations of ethanol equal to and greater than 3% (vol/vol) also inhibited this enzyme in vitro. The possible role of the in vivo activation of the plasma membrane proton-pumping ATPase in the development of ethanol tolerance by this fermenting yeast was discussed.     

In vivo activation by ethanol of plasma membrane ATPase of Saccharomyces cerevisiae.

Ethanol, in concentrations that affect growth and fermentation rates (3 to 10% [vol/vol]), activated in vivo the plasma membrane ATPase of Saccharomyces cerevisiae. The maximal value for this activated enzyme in cells grown with 6 to 8% (vol/vol) ethanol was three times higher than the basal level (in cells grown in the absence of ethanol). The Km values for ATP, the pH profiles, and the sensitivities to orthovanadate of the activated and the basal plasma membrane ATPases were virtually identical. A near-equivalent activation was also observed when cells grown in the absence of ethanol were incubated for 15 min in the growth medium with ethanol. The activated state was preserved after the extraction from the cells of the membrane fraction, and cycloheximide appeared to prevent this in vivo activation. After ethanol removal, the rapid in vivo reversion of ATPase activation was observed. While inducing the in vivo activation of plasma membrane ATPase, concentrations of ethanol equal to and greater than 3% (vol/vol) also inhibited this enzyme in vitro. The possible role of the in vivo activation of the plasma membrane proton-pumping ATPase in the development of ethanol tolerance by this fermenting yeast was discussed.     

13C NMR studies of butyric fermentation in Clostridium kluyveri

The fermentation of 13C-labeled ethanol and acetate into butyrate and caproate by Clostridium kluyveri has been studied by using 13C NMR. The pathway involves the conversion of both ethanol and acetate into acetyl coenzymes A, two of which condense to form CoA-linked precursors of butyrate. If butyryl-CoA is involved in the condensation, caproate is the ultimate product. ATP is produced from acetyl-CoA via the reactions catalyzed by phosphotransacetylase and acetate kinase with acetate, a required carbon source, as a co-product. In spectra of whole cells incubated with the labeled carbon sources, label from ethanol appears rapidly in acetate, which then reaches a lower, steady-state concentration due to its re-entry into the pathway. The rapid initial production of acetate indicates equally rapid production of ATP. Label from acetate appears in ethanol only if ethanol is already present, indicating that this process is one of isotopic equilibration rather than net synthesis of ethanol from acetate. The ratio of butyrate to caproate produced depends strongly on the initial ratio of ethanol to acetate in the medium. The relative rates of utilization of ethanol and acetate vary as the fermentation proceeds. 13C-13C coupling in the butyrate and caproate produced from [1-13C]ethanol and [2-13C]acetate can be used to determine if the acetyl-CoA molecules arising from ethanol and acetate enter the same pool or if they remain separated. The data are consistent with random mixing of the acetyl-CoA produced from the two carbon sources.     

Inhibition of succinate production during yeast fermentation by deenergization of the plasma membrane

Succinate production by a respiratory-deficient yeast was inibited by substances known to depolarize the plasma membrane. These substances include high concentrations of the permeable cation potassium, the ATPase inhibitor diethylstilbestrol, the polyene antibiotic amphotericin B, and the uncouplers carbonyl cyanidem-chlorophenylhydrazone and dinitrophenol. Results suggest that succinate effux from yeast cells is driven by membrane energization in the form of an electrical potential. As sucinate is one of the major by-products of alcoholic fermentation, deenergization of yeast plasma membrane may be a useful approach to increasing the yield of ethanol in industrial fermentations.     

Expression of Cellulose Synthase Genes During the Gravistimulation of Flax (Linum usitatissimum) and Poplar (Populus alba × tremula) Plants

Russian Journal of Bioorganic Chemistry - Plant cellulose is synthesized on the plasma membrane by the cellulose synthase complex and a number of coenzymes. Different cellulose synthases are...     

Membrane Fluidity Adjustments in Ethanol-Stressed Oenococcus oeni Cells

The effect of ethanol on the cytoplasmic membrane of Oenococcus oeni cells and the role of membrane changes in the acquired tolerance to ethanol were investigated. Membrane tolerance to ethanol was defined as the resistance to ethanol-induced leakage of preloaded carboxyfluorescein (cF) from cells. To probe the fluidity of the cytoplasmic membrane, intact cells were labeled with doxyl-stearic acids and analyzed by electron spin resonance spectroscopy. Although the effect of ethanol was noticeable across the width of the membrane, we focused on fluidity changes at the lipid-water interface. Fluidity increased with increasing concentrations of ethanol. Cells responded to growth in the presence of 8% (vol/vol) ethanol by decreasing fluidity. Upon exposure to a range of ethanol concentrations, these adapted cells had reduced fluidity and cF leakage compared with cells grown in the absence of ethanol. Analysis of the membrane composition revealed an increase in the degree of fatty acid unsaturation and a decrease in the total amount of lipids in the cells grown in the presence of 8% (vol/vol) ethanol. Preexposure for 2 h to 12% (vol/vol) ethanol also reduced membrane fluidity and cF leakage. This short-term adaptation was not prevented in the presence of chloramphenicol, suggesting that de novo protein synthesis was not involved. We found a strong correlation between fluidity and cF leakage for all treatments and alcohol concentrations tested. We propose that the protective effect of growth in the presence of ethanol is, to a large extent, based on modification of the physicochemical state of the membrane, i.e., cells adjust their membrane permeability by decreasing fluidity at the lipid-water interface.     

Membrane fluidity adjustments in ethanol-stressed Oenococcus oeni cells

The effect of ethanol on the cytoplasmic membrane of Oenococcus oeni cells and the role of membrane changes in the acquired tolerance to ethanol were investigated. Membrane tolerance to ethanol was defined as the resistance to ethanol-induced leakage of preloaded carboxyfluorescein (cF) from cells. To probe the fluidity of the cytoplasmic membrane, intact cells were labeled with doxyl-stearic acids and analyzed by electron spin resonance spectroscopy. Although the effect of ethanol was noticeable across the width of the membrane, we focused on fluidity changes at the lipid-water interface. Fluidity increased with increasing concentrations of ethanol. Cells responded to growth in the presence of 8% (vol/vol) ethanol by decreasing fluidity. Upon exposure to a range of ethanol concentrations, these adapted cells had reduced fluidity and cF leakage compared with cells grown in the absence of ethanol. Analysis of the membrane composition revealed an increase in the degree of fatty acid unsaturation and a decrease in the total amount of lipids in the cells grown in the presence of 8% (vol/vol) ethanol. Preexposure for 2 h to 12% (vol/vol) ethanol also reduced membrane fluidity and cF leakage. This short-term adaptation was not prevented in the presence of chloramphenicol, suggesting that de novo protein synthesis was not involved. We found a strong correlation between fluidity and cF leakage for all treatments and alcohol concentrations tested. We propose that the protective effect of growth in the presence of ethanol is, to a large extent, based on modification of the physicochemical state of the membrane, i.e., cells adjust their membrane permeability by decreasing fluidity at the lipid-water interface.     

A study of ethanol tolerance in yeast

The ethanol tolerance of yeast and other microorganisms has remained a controversial area despite the many years of study. The complex inhibition mechanism of ethanol and the lack of a universally accepted definition and method to measure ethanol tolerance have been prime reasons for the controversy. A number of factors such as plasma membrane composition, media composition, mode of substrate feeding, osmotic pressure, temperature, intracellular ethanol accumulation, and byproduct formation have been shown to influence the ethanol tolerance of yeast. Media composition was found to have a profound effect upon the ability of a yeast strain to ferment concentrated substrates (high osmotic pressure) and to ferment at higher temperatures. Supplementation with peptone-yeast extract, magnesium, or potassium salts has a significant and positive effect upon overall fermentation rates. An intracellular accumulation of ethanol was observed during the early stages of fermentation. As fermentation proceeds, the intracellular and extracellular ethanol concentrations become similar. In addition, increases in osmotic pressure are associated with increased intracellular accumulation of ethanol. However, it was observed that nutrient limitation, not increased intracellular accumulation of ethanol, is responsible to some extent for the decreases in growth and fermentation activity of yeast cells at higher osmotic pressure and temperature.     

The regeneration of coenzymes using immobilized hydrogenase

Summary A new enzymatic approach to the regeneration of coenzymes has been developed. It uses the reduction of natural or artificial cofactors with H₂, catalyzed by hydrogenase activity in immobilized whole cells ofA. eutrophus. The method has been employed for regeneration of such coenzymes as NAD⁺, FMN, phenazine methosulfate, Janus green, methylene blue, and 2,6-dichlorophenol-indophenol.     

Influence of metal addition on ethanol production with <Emphasis Type="Italic">Pichia stipitis</Emphasis> ATCC 58784

Trace metals always act as cofactors or coenzymes in many cellular processes. Deficiency or excess of some metals will affect the fermentation of lignocellulosic hydrolysate. In order to make sure the deficient or excessive states of metals in culture medium, metal contents analysis was conducted in Pichia stipitis ATCC 58784 cells, synthetic medium, and diluted acid hydrolysate of rice straw. The results showed that Cu, Ni, and Co were deficient, and Al was a little excessive. So the influences of Cu²⁺, Al³⁺, Ni²⁺, and Co²⁺ additions on the growth and ethanol production of ATCC 58784 were further researched. Low concentration additions of Cu²⁺ and Al³⁺ (<0.24 mM and <0.23 mM, respectively) improved biomass growth of ATCC 58784 by 34 and 13%, respectively; however, higher concentrations decreased biomass growth. On the other hand, addition of Cu²⁺ (0.39 mM) did not affect volumetric ethanol production significantly (P = 0.05) and addition of Al³⁺ (0.38 mM) showed no influence on volumetric ethanol production (P = 0.68). Addition of 0.074 mM Co²⁺ inhibited biomass growth of ATCC 58784 by 13% and volumetric ethanol production by 10%. The biomass growth and volumetric ethanol production of ATCC 58784 was arrested by the addition of 0.33 mM of Ni²⁺ by 53 and 65%, respectively.     

The speed-controlling reactions in fermentation of quickly dried yeast

Several factors which influence the speed of fermentation of quickly dried yeast are investigated. If the yeast is washed and the bulk of coenzymes and phosphate is removed, addition of 0.5 μmole diphosphopyridine nucleotide (DPN) and 0.5 μmole adenosine triphosphate (ATP) per cc. is necessary for maximal speed. In the optimal pH range, which lies between 6.6 and 6.2, and with optimal amounts of cofactors, there is no influence of nicotinamide mononucleotide (NMN), but with suboptimal amounts of DPN, the speed is raised by synthesis to DPN.The dialyzate of boiled juice contains factors which raise the speed of washed yeast by 20–30% of the maximum obtained in the presence of the usual cofactors. A phosphate concentration of 0.01–0.02 M is likewise necessary even if the phosphate is only partly esterified. At pH 6.0 to 5.9 the speed is less than half that at pH 6.5.Fermentation is completely absent without either K⁺ or NH₄⁺ and without Mg. The optimal amount of the monovalent ions is 5 × 10^?2M. Sodium alone is unable to allow fermentation but is only slightly harmful if enough K⁺ or NH₄⁺ is present.Addition of small amounts of phosphoglycerate at optimal potassium phosphate concentration and pH increases the rate of sugar fermentation and gives rise to an extra CO₂ formation during the time of phosphoglycerate decomposition of about 3 to 5 times the amount added.     

Continuous ethanol fermentation of concentrated sugar solutions coupled with membrane distillation using a PTFE module

Continuous ethanol fermentation of concentrated glucose and molasses solutions was coupled with membrane distillation using a PTFE ethanol stripping module. Experimental results indicated that the PTFE module can remove a high concentration of ethanol from the fermentation broth and thus maintain a low ethanol concentration in the broth, thereby alleviating the problem of product inhibition. Accordingly, the product yield and the specific ethanol production rate increased. During the continuous fermentation runs, long-time operation using the PTFE module was found to be possible (i.e. 430 h using the glucose medium and 695 h using the molasses medium) and no significant change in the ethanol separation performance of the PTFE module was observed. Although cell flocculation became undetectable when a concentrated molasses medium containing 316 g/l sugar solution was used, the ethanol separation performance of the ethanol stripper was not adversely affected by the presence of the free cells. This suggests that clogging of the membrane pores by cells or other particulates is not a major problem when using the PTFE module in continuous ethanol fermentation.     

Effect of ethanol on glucose transport, key glycolytic enzymes, and proton extrusion in Saccharomyces cerevisiae

The effect of ethanol on the activities of the key enzymes of the glycolytic pathway and on two membrane functions related with fermentation, the glucose uptake system, and proton extrusion rate are examined. The results indicate that ethanol, up to 2M, does not cause any change of the glucose uptake velocity nor any substantial change in the key glycolytic enzyme activities while the fermentation rate is reduced by about 50%. In a cell extract 3M ethanol as well as incubation of yeast cells with 4M ethanol caused a considerable decrease of pyruvate kinase and hexokinase activities. Phosphofructokinase remained unchanged even at higher ethanol concentrations. Transmembrane proton flow was found to be the most sensitive of the functions tested toward ethanol, and it could represent the first target of ethanol action on fermentation.     

Intracellular acidification does not account for inhibition of Saccharomyces cerevisiae growth in the presence of ethanol

Abstract Intracellular acidification has been considered one of a number of mechanisms underlying the inhibition of growth and fermentation by ethanol in yeast. However, most of the studies on the effect of ethanol on yeast intracellular pH (pH_i) were carried out by using unadapted cells to which ethanol was added. In this paper we show that the pH_i of exponential cells of Saccharomyces cerevisiae IGC 3507 III grown in a medium with glucose and inhibitory concentrations of ethanol only decreased to values below those in unstressed cells (6.9) for concentrations equal to or above 7% (v/v). Only at these supracritical levels (7–10% (v/v)) was pH homeostasis in ethanol-adapted yeast affected. This is consistent with the significant increase of plasma membrane permeability and decrease of plasma membrane H⁺-ATPase in comparison with the corresponding values in unstressed cells. These deleterious effects were only observed with those high concentrations of toxin. These results indicate that intracellular acidification does not account for inhibition of yeast growth in the presence of ethanol. In fact, growth was inhibited by ethanol concentrations (3–6% (v/v)) that did not lead to the decrease of pH_i. Furthermore, even for supracritical concentrations, close to the maximal that allowed growth (10% (v/v)), the dedrease of pH_i was not important reaching, at the most, values of 6.5–6.6.     

Radiation Damage to the Erythrocyte Membrane: The Effects of Medium and Cell Concentrations

Human erythrocytes suspended in plasma, or in phosphate buffered saline (PBS), were exposed to ionizing radiation. Potassium leakage from irradiated erythrocytes is significantly higher in PBS than in plasma. The potassium leakage decreases when PBS is gradually replaced by plasma. These findings suggest that some of the plasma constituents have radioprotective properties. The potassium leakage per cell is independent of the hematocrit, Hct. The potassium leakage is attributed to the formation of radiation defects in the membrane. Analysis of the effect of radiation dose, plasma and cell concentrations on the product of the number and surface area of the radiation defects indicates that the radiation damage is mainly due to the direct formation of free radicals in the cell membrane. The radioprotective effect of plasma is attributed to surface reactions of these free radicals with plasma constituents adsorbed on the membrane.     

Radiation Damage to the Erythrocyte Membrane: The Effects of Medium and Cell Concentrations

Human erythrocytes suspended in plasma, or in phosphate buffered saline (PBS), were exposed to ionizing radiation. Potassium leakage from irradiated erythrocytes is significantly higher in PBS than in plasma. The potassium leakage decreases when PBS is gradually replaced by plasma. These findings suggest that some of the plasma constituents have radioprotective properties. The potassium leakage per cell is independent of the hematocrit, Hct. The potassium leakage is attributed to the formation of radiation defects in the membrane. Analysis of the effect of radiation dose, plasma and cell concentrations on the product of the number and surface area of the radiation defects indicates that the radiation damage is mainly due to the direct formation of free radicals in the cell membrane. The radioprotective effect of plasma is attributed to surface reactions of these free radicals with plasma constituents adsorbed on the membrane.     

Effects of alcohols on the respiration and fermentation of aerated suspensions of baker's yeast.

The immediate effects of externally added alcohols on CO2 production and O2 consumption of suspensions of washed, aerated baker's yeast were studied by stopped-flow membrane inlet mass spectrometry. Glucose-supported fermentation was progressively inhibited by increasing ethanol concentration (0-20%, v/v). The inhibition by ethanol was quite different from that observed for acetaldehyde; thus it is unlikely that toxicity of the latter can account for the observed effects. For five different alkanols (methanol, ethanol, 1-propanol, 2-propanol and 1-butanol) increasing inhibition of anaerobic fermentation was correlated with increased partition coefficients into a hydrophobic milieu. This suggests that the action of ethanol is primarily located at a hydrophobic site, possibly at a membrane. Results for respiratory activities were not as definite as for those for anaerobic metabolism because some alkanols act as respiratory substrates as well as giving inhibitory effects.     

The susceptibility of membrane vesicles to interaction with ethanol

The susceptibility of membranes to interaction with ethanol is an important consideration in the further understanding of the ethanol-membrane interaction. Interaction of membrane vesicles, including passive diffusion of ethanol across membranes, leakage of internal molecules out of membranes and membrane-membrane interaction, were examined systematically using two populations of fluorescent probe-encapsulated phospholipid bilayer vesicles, each prepared with 1,2-dimyristoyl phosphatidylcholine, cholesterol and a fluorescent probe. Fluorescence quenching experiments with these vesicles were performed in a medium containing a wide range of ethanol concentrations (0.30-3.5 M). In the presence of a lower concentration of ethanol in the external medium, passive diffusion of ethanol across membrane vesicles occurred. This was demonstrated by an interaction of ethanol with the encapsulated fluorescence probe molecules inside the vesicles, resulting in an increase in the fluorescence intensity and a shift of the fluorescence emission spectrum to a shorter wavelength. While, in the presence of a higher concentration of ethanol in the external medium, a strong perturbation of lipid bilayers by ethanol was found, leading to an over expansion of membranes and consequently causing the membrane leakage. As a result of this, the initially encapsulated probe molecules leaked out of the vesicles so as to interact with the other probe molecules in the external medium. Consequently, fluorescence quenching was observed. Moreover, studies of the mixture of two populations of fluorescence probe-encapsulated membrane vesicles revealed that ethanol acted on individual membranes and did not promote membrane-membrane interactions. The implication of the present results to the alcohol-mediated expansion of membranes is discussed.