Effects of Ethanol and Other Alkanols on Transport of Acetic Acid in Saccharomyces cerevisiae

In glucose-grown cells of Saccharomyces cerevisiae IGC 4072, acetic acid enters only by simple diffusion of the undissociated acid. In these cells, ethanol and other alkanols enhanced the passive influx of labelled acetic acid. The influx of the acid followed first-order kinetics with a rate constant that increased exponentially with the alcohol concentration, and an exponential enhancement constant for each alkanol was estimated. The intracellular concentration of labelled acetic acid was also enhanced by alkanols, and the effect increased exponentially with alcohol concentration. Acetic acid is transported across the plasma membrane of acetic acid-, lactic acid-, and ethanol-grown cells by acetate-proton symports. We found that in these cells ethanol and butanol inhibited the transport of labelled acetic acid in a noncompetitive way; the maximum transport velocity decreased with alcohol concentration, while the affinity of the system for acetate was not significantly affected by the alcohol. Semilog plots of V_max versus alcohol concentration yielded straight lines with negative slopes from which estimates of the inhibition constant for each alkanol could be obtained. The intracellular concentration of labelled acid was significantly reduced in the presence of ethanol or butanol, and the effect increased with the alcohol concentration. We postulate that the absence of an operational carrier for acetate in glucose-grown cells of S. cerevisiae, combined with the relatively high permeability of the plasma membrane for the undissociated acid and the inability of the organism to metabolize acetic acid, could be one of the reasons why this species exhibits low tolerance to acidic environments containing ethanol.     

A peculiar behaviour for cell death induced by weak carboxylic acids in the wine spoilage yeast Zygosaccharomyces bailii

In glucose-grown cells of Zygosaccharomyces bailii , ISA 1307 acetic acid and other carboxylic acids enhanced death. The effects were much lower than those described for Saccharomyces cerevisiae , being only detectable at higher acid concentrations. In Z. bailii, acetic acid and other weak acids also induced intracellular acidification, but this effect was less pronounced than that of death and no relationship was found with death enhancement. The results suggested that in Z. bailii , unlike S. cerevisiae , intracellular acidification induced by weak acids is less pronounced and appears not to have a significant role in death at the temperature range used.     

Effect of external pH on acidification and excretion of ethanol intermediates byCandida utilis

External pH affects the acidification induced by ethanol. The apparent specific rate of acidification depends on the dissociation properties and production of carbon dioxide, acetic and lactic acid which are intermediates of ethanol oxidation. The organic acids are transported by Nernst — Einstein diffusion. We designed and identified a new and simple mathematical model that allows us to describe the effect of external pH on nonstationary transport of dissociated intermediates of ethanol oxidation.     

Transport of malic acid and other dicarboxylic acids in the yeast Hansenula anomala

DL-Malic acid-grown cells of the yeast Hansenula anomala formed a saturable transport system that mediated accumulative transport of L-malic acid with the following kinetic parameters at pH 5.0: Vmax, 0.20 nmol.s-1.mg (dry weight)-1; Km, 0.076 mM L-malate. Uptake of malic acid was accompanied by proton disappearance from the external medium with rates that followed Michaelis-Menten kinetics as a function of malic acid concentration. Fumaric acid, alpha-ketoglutaric acid, oxaloacetic acid, D-malic acid, and L-malic acid were competitive inhibitors of succinic acid transport, and all induced proton movements that followed Michaelis-Menten kinetics, suggesting that all of these dicarboxylates used the same transport system. Maleic acid, malonic acid, oxalic acid, and L-(+)-tartaric acid, as well as other Krebs cycle acids such as citric and isocitric acids, were not accepted by the malate transport system. Km measurements as a function of pH suggested that the anionic forms of the acids were transported by an accumulative dicarboxylate proton symporter. The accumulation ratio at pH 5.0 was about 40. The malate system was inducible and was subject to glucose repression. Undissociated succinic acid entered the cells slowly by simple diffusion. The permeability of the cells by undissociated acid increased with pH, with the diffusion constant increasing 100-fold between pH 3.0 and 6.0.     

Transport of malic acid and other dicarboxylic acids in the yeast Hansenula anomala.

DL-Malic acid-grown cells of the yeast Hansenula anomala formed a saturable transport system that mediated accumulative transport of L-malic acid with the following kinetic parameters at pH 5.0: Vmax, 0.20 nmol.s-1.mg (dry weight)-1; Km, 0.076 mM L-malate. Uptake of malic acid was accompanied by proton disappearance from the external medium with rates that followed Michaelis-Menten kinetics as a function of malic acid concentration. Fumaric acid, alpha-ketoglutaric acid, oxaloacetic acid, D-malic acid, and L-malic acid were competitive inhibitors of succinic acid transport, and all induced proton movements that followed Michaelis-Menten kinetics, suggesting that all of these dicarboxylates used the same transport system. Maleic acid, malonic acid, oxalic acid, and L-(+)-tartaric acid, as well as other Krebs cycle acids such as citric and isocitric acids, were not accepted by the malate transport system. Km measurements as a function of pH suggested that the anionic forms of the acids were transported by an accumulative dicarboxylate proton symporter. The accumulation ratio at pH 5.0 was about 40. The malate system was inducible and was subject to glucose repression. Undissociated succinic acid entered the cells slowly by simple diffusion. The permeability of the cells by undissociated acid increased with pH, with the diffusion constant increasing 100-fold between pH 3.0 and 6.0.     

Individual cells of Saccharomyces cerevisiae and Zygosaccharomyces bailii exhibit different short-term intracellular pH responses to acetic acid

The effects of perfusion with 2.7 and 26 mM undissociated acetic acid in the absence or presence of glucose on short-term intracellular pH (pH(i)) changes in individual Saccharormyces cerevisiae and Zygosaccharomyces bailii cells were studied using fluorescence-ratio-imaging microscopy and a perfusion system. In the S. cerevisiae cells, perfusion with acetic acid induced strong short-term pH(i) responses, which were dependent on the undissociated acetic acid concentration and the presence of glucose in the perfusion solutions. In the Z. bailii cells, perfusion with acetic acid induced only very weak short-term pH(i) responses, which were neither dependent on the undissociated acetic acid concentration nor on the presence of glucose in the perfusion solutions. These results clearly show that Z. bailii is more resistant than S. cerevisiae to short-term pH(i) changes caused by acetic acid.     

Glucose respiration and fermentation in Zygosaccharomyces bailii and Saccharomyces cerevisiae express different sensitivity patterns to ethanol and acetic acid

In the yeast Zygosaccharomyces bailii ISA 1307, respiration and fermentation ofglucose were exponentially inhibited by ethanol, both processes displaying similar sensitivity tothe alcohol. Moreover, the degree of inhibition on fermentation was of the same magnitude as thatreported for Saccharomyces cerevisiae. Acetic acid also inhibited these two metabolicprocesses in Z. bailii , with the kinetics of inhibition again being exponential. However,inhibition of fermentation was much less pronounced than in S. cerevisiae. The valuesestimated with Z. bailii for the minimum inhibitory concentration of acetic acid rangedfrom 100 to 240 mmol l⁻¹ total acetic acid compared with values of near zeroreported for S. cerevisiae. The inhibitory effects of acetic acid on Z. bailii were notsignificantly potentiated by ethanol.     

Resistance of some wine spoilage yeasts to combinations of ethanol and acids present in wine

The most important factors affecting microbial growth in an alcoholic beverage are the ethanol content and the low pH. The effectiveness of different organic acids in conjunction with ethanol concentration in controlling growth of yeasts was determined for Saccharomyces cerevisiae, Schizosaccharomyces pombe, Brettanomyces lambicus, Pichia anomala, Zygosaccharomyces bailii, Saccharomycodes ludwigii and Kluyveromyces thermotolerans in malt extract broth (MEB). The results are summarized as undissociated concentrations of different acids required to inhibit growth of yeasts in MEB containing 10% (v/v) ethanol. About half the amount of undissociated malic or tartaric acid is necessary for inhibition of the yeasts, compared with acetic and lactic acid but the concentrations of acid necessary to inhibit growth were generally very high and unrealistic in wines for controlling growth of most of the yeasts tested. All the yeasts tested were able to grow in acidified non- or low alcoholic beverages but, at higher ethanol concentrations, Sacch. cerevisiae and Zygosacch. bailii have the greatest spoilage potential.     

Effects of ethanol and acetic acid on the transport of malic acid and glucose in the yeast Schizosaccharomyces pombe: implications in wine deacidification

Abstract Ethanol and acetic acid, at concentrations which may occur during wine-making, inhibited the transport of l-malic acid in Schizosaccharomyces pombe . The inhibition was non-competitive, the decrease of the maximum initial velocity following exponential kinetics. Glucose transport was not significantly affected either by ethanol (up to 13%, w/v) or by acetic acid (up to 1.5%, w/v). The uptake of labelled acetic acid followed simple diffusion kinetics, indicating that a carrier was not involved in its transport. Therefore, the undissociated acid appears to be the only form that enters the cells and is probably responsible for the toxic effects. Accordingly, deacidification by Ss. pombe during wine fermentation should take place before, rather than after, the main alcoholic fermentation by Saccharomyces cerevisiae .     

Adaptive response to acetic acid in the highly resistant yeast species Zygosaccharomyces bailii revealed by quantitative proteomics

Zygosaccharomyces bailii is the most tolerant yeast species to acetic acid-induced toxicity, being able to grow in the presence of concentrations of this food preservative close to the legal limits. For this reason, Z. bailii is the most important microbial contaminant of acidic food products but the mechanisms behind this intrinsic resistance to acetic acid are very poorly characterized. To gain insights into the adaptive response and tolerance to acetic acid in Z. bailii, we explored an expression proteomics approach, based on quantitative 2DE, to identify alterations occurring in the protein content in response to sudden exposure or balanced growth in the presence of an inhibitory but nonlethal concentration of this weak acid. A coordinate increase in the content of proteins involved in cellular metabolism, in particular, in carbohydrate metabolism (Mdh1p, Aco1p, Cit1p, Idh2p, and Lpd1p) and energy generation (Atp1p and Atp2p), as well as in general and oxidative stress response (Sod2p, Dak2p, Omp2p) was registered. Results reinforce the concept that glucose and acetic acid are coconsumed in Z. bailii, with acetate being channeled into the tricarboxylic acid cycle. When acetic acid is the sole carbon source, results suggest the activation of gluconeogenic and pentose phosphate pathways, based on the increased content of several proteins of these pathways after glucose exhaustion.     

Influence of ethanol and temperature on the cellular fatty acid composition of Zygosaccharomyces bailii spoilage yeasts

Changes in the fatty acid profile of Zygosaccharomyces bailii strains, isolated from different sources, after growth at increasing concentrations of ethanol and/or decreasing temperatures were determined. Differences in fatty acid composition between Zygosaccharomyces bailii strains at standard conditions (25°C, 0% initial ethanol) were observed and could be related to ethanol tolerance. Zygosaccharomyces bailii strain isolated from wine showed the highest ethanol tolerance in relation to growth rate. Surprisingly, an increase in ethanol concentration or a decrease in growth temperature caused a decrease in the degree of unsaturation of total cellular fatty acids. On the other hand, the mean chain length increased (high ethanol concentration) or decreased (low temperature) depending on the stress factor. When both stress situations (high ethanol concentration and low temperature) were present at the same time, the degree of unsaturation remained approximately constant. With decreasing temperatures, the C16/C18 ratio increased in studies of initial ethanol content below 5%, and above 5% ethanol, decreased.     

Incorporation of 14C-amino acids by malarial plasmodia (Plasmodium iophurae). VI. Changes in the kinetic constants of amino acid transport during infection

The majority (10 of 17) of amino acids tested entered the mature duck erythrocyte by a saturable, non-uphill transport system, whereas for the erythrocyte-free malarial parasite, Plasmodium lophurae, the converse was true: most amino acids entered the parasite by simple diffusion. Only five amino acids (glutamic and aspartic acids, cysteine, lysine, arginine) showed mediated entry into P. lophurae. The pattern of mediated amino acid transport into the duck erythrocyte was altered upon infection, e.g., either entry was by diffusion or there was a reduced affinity for the amino acid. Transport characteristics similar to those found in the malaria-infected erythrocyte were produced by treating normal duck red cells with a cell-free extract of malaria-infected erythrocytes and quinine (a depressor of red cell ATP). It is suggested that depletion of host cell ATP as well as elaboration of as yet unidentified substances by the parasite promote the changes in permeability seen in the malaria-infected cell.     

Initiation of Yeast Sporulation by Partial Carbon, Nitrogen, or Phosphate Deprivation

In this paper we show that partial deprivation of a carbon source, a nitrogen source, or phosphate in the presence of all other nutrients needed for growth initiates meiosis and sporulation of Saccharomyces cerevisiae homothallic strain Y55. For carbon deprivation experiments, cells were grown in synthetic medium (pH 5.5) containing an excess of one carbon source and then transferred to the same medium containing different concentrations of the same carbon source. In the case of transfer to different acetate concentrations, the log optical density at 600 nm increased at the previous rate until the cells had used up all of the acetate, whereupon the cells entered a stationary phase and did not sporulate. The same was observed with ethanol. In contrast, at different concentrations of dihydroxy-acetone or pyruvate, cells grew at different rates and sporulated optimally at intermediate concentrations (50 to 75 mM). The response to galactose was similar but reflected the presence of a low-affinity galactose transport system and the induction of a high-affinity galactose transport system. Cells could also sporulate when a glucose medium ran out of glucose, apparently because they initiated sporulation during the subsequent lag period and then used the produced ethanol as a carbon source. For phosphate deprivation experiments, cells growing with excess ethanol or pyruvate and phosphate were transferred to the same medium containing limiting amounts of phosphate. First, they used up the intracellular phosphate reserves for rapid growth, and then they sporulated optimally when an intermediate concentration (30 μM) of phosphate had been added to the medium. For nitrogen deprivation experiments, cells grown with excess acetate, ethanol, or pyruvate and NH₄⁺ were transferred to the same medium from which all nitrogen had been removed. These cells sporulated well in acetate medium but poorly in ethanol and pyruvate media. However, the sporulation frequency in the latter media could be increased greatly by adding intermediate concentrations (1 mM) of the slowly metabolizable amino acids glycine, histidine, or phenylalanine. If one assumes that the sporulation response to partial deprivation of carbon-, nitrogen-, or phosphorus-containing compounds reflects control by a single metabolite, the intracellular concentration of this metabolite may decide at the START position (G1 phase) of the cell cycle whether a/α cells enter mitosis or meiosis.     

Mechanism of Resistance of Saccharomyces bailii to Benzoic, Sorbic and Other Weak Acids Used as Food Preservatives

Saccharomyces bailii grows in the presence of high concentrations of sorbic, benzoic and other short-chain monocarboxylic acids commonly used as preservatives. Starved cells concentrate these acids intracellularly, approximately as expected from the pH of the ceil and the p K _a of the acid. On addition of glucose, the intracellular content of preservative is considerably reduced. The glucose effect is sensitive to metabolic inhibitors, and anaerobic respiration is stimulated by the preservatives. The ability to maintain a low intracellular concentration of any of the preservatives tested is induced by growth in the presence of sorbic or benzoic acid and less effectively by butyric or acetic acid. Both induced and uninduced cells are permeable to benzoic and butyric acids. Benzoate and sorbate are not metabolized at a rate significant with respect to the permeation rate. Resistance to these preservatives apparently results primarily from an inducible, energy requiring system which transports preservative from the cell.     

Comparative physiologial studies of the yeast and mycelial forms of Histoplasma capsulaum: uptake and incorporation of L-leucine

l-Leucine entered the cells of both morphological forms of Histoplasma capsulatum by a permease-like system at low external concentrations of substrate. However, at levels greater than 5 x 10(-5)m l-leucine, the amino acid entered the cells both through a simple diffusion-like process and the permease-like system. The rate of the amino acid diffusion into yeast and mycelial forms appeared to be the same, whereas the initial rate of accumulation through the permease-like system was 5 to 10 times faster in the mycelial phase than it was in the yeast phase. The Michaelis constants were 2.2 x 10(-5)m in yeast phase and 2 x 10(-5)m in mycelial phase cells. Transport of l-leucine at an external concentration of 10(-5)m showed all of the characteristics of a system of active transport, which was dependent on temperature and pH. Displacement or removal of the alpha-amino group, or modification of the alpha-carboxyl group abolished amino acid uptake. The process was competitively inhibited by neutral aliphatic side-chain amino acids (inhibition constants ranged from 1.5 x 10(-5) to 6.2 x 10(-5)m). Neutral aromatic side-chain amino acids and the d-isomers of leucine and valine did not inhibit l-leucine uptake. These data were interpreted to mean that the l-leucine transport system is stereospecific and is highly specific for neutral aliphatic side-chain amino acids. Incorporation of l-leucine into macromolecules occurred at almost the same rate in both morphological forms of the fungus. The mycelial phase but not the yeast phase showed a slight initial lag in incorporation. In both morphological forms the intracellular pool of l-leucine was of limited capacity, and the total uptake of the amino acid was a function of intracellular pool size. The initial rate of l-leucine uptake was independent of the level of intracellular pool. Both morphological forms deaminated and degraded only a minor fraction of the accumulated leucine.     

Nutrition of Acetobacter rancens S3 and F11 Isolated from Tanks for Vinegar Production

The strains S3 and F11 which were isolated respectively from static and submerged tanks for vinegar production were identified as Acetobacter rancens. Neither strain grew in an ammonium defined medium containing ethanol, glucose, glycerol or organic acids as the sole carbon source. When casamino acids were added, they grew luxuriantly with lactate, ethanol or glycerol as the carbon source and less well with acetate or glucose. They grew, forming much acetic acid, in defined ethanol medium when alanine was supplied in place of casamino acids, but strain S3 showed a longer lag time than strain Fl1. This lag time could be shortened by addition of aspartate and glutamate. These amino acids could be replaced by succinate, fumarate, malate, lactate, pyruvate or propionate but not by glucose. Both strains required lactate or pyruvate in defined glucose medium but many other organic acids, which were effective in defined ethanol medium, were ineffective or slightly effective in glucose medium.     

Aerobic sugar metabolism in the spoilage yeast Zygosaccharomyces bailii

Despite the importance of some Zygosaccharomyces species as agents causing spoilage of food, the carbon and energy metabolism of most of them is yet largely unknown. This is the case with Zygosaccharomyces bailii. In this study the occurrence of the Crabtree effect in the petite-negative yeast Z. bailii ATCC 36947 was investigated. In this yeast the aerobic ethanol production is strictly dependent on the carbon source utilised. In glucose-limited continuous cultures a very low level of ethanol was produced. In fructose-limited continuous cultures ethanol was produced at a higher level and its production increased with the dilution rate. As a consequence, on fructose the onset of respiro-fermentative metabolism caused a reduction in biomass yield. An immediate aerobic alcoholic fermentation in Z. bailii was observed during the transition from sugar limitation to sugar excess, both on glucose and on fructose. The analysis of some key enzymes of the fermentative metabolism showed a high level of acetyl-CoA synthetase in Z. bailii growing on fructose. At high dilution rates, the activities of glucose- and fructose-phosphorylating enzymes, as well as of pyruvate decarboxylase and alcohol dehydrogenase, were higher in cells during growth on fructose than on glucose.     

Presence of proline has a protective effect on weak acid stressed Saccharomyces cerevisiae

Fermentation of sugars released from lignocellulosic biomass (LCMs) is a sustainable option for the production of bioethanol. LCMs release fermentable hexose sugars and the currently non-fermentable pentose sugars; ethanol yield from lignocellulosic residues is dependent on the efficient conversion of available sugars to ethanol, a side-product of the process is acetic acid production. Presence of acetic acid reduced metabolic output and growth when compared with controls; however, it was observed that incubation with proline had a protective effect, which was proline specific and concentration dependent; the protective effect did not extend to furan or phenolic stressed yeast cells. Proline accumulating strains displayed tolerance to acetic acid when compared with background strains, whereas, strains with a compromised proline metabolism displayed sensitivity. Sensitivity to weak acids appears to be reduced with the addition of proline; proline is an imino acid freely available as a nitrogen source in the aerobic phase of fermentations. Yeast strains with higher intracellular proline concentrations would be desirable for industrial bioethanol fermentations.     

胞内氧化还原水平对嗜热厌氧乙醇菌 发酵代谢的影响

辅酶NADH/NAD+在细胞内氧化还原反应中起着重要的作用,是细胞生长和能量代谢必不可少的辅因子。调节微生物胞内NADH/NAD+的比率是定向改变微生物代谢,高效获得目标代谢产物的有效手段。嗜热厌氧乙醇菌(Thermoanaerobacter ethanolicus)是高温厌氧菌中乙醇产量较高的代表性菌株,本文利用不同氧化还原态的碳源改变T.ethanolicus的胞内NADH/NAD+含量和比例,进而研究了其对细胞生长、代谢产物分布的影响。以不同比例的葡萄糖/甘露醇作为混合碳源发酵,胞内氧化还原水平、细胞的生长特性、代谢产物都发生了不同程度的差异,以葡萄糖作为唯一碳源进行培养时,T.ethanolicus生长良好,乙醇产量为0.79g/L,但胞内NADH/NAD+比值和乙醇/乙酸的比值都比较低,分别为0.47和4.82;随着葡萄糖在混合碳源中比例的下降,NADH/NAD+比值增高,发酵产物中乙醇/乙酸比值也呈现上升的趋势。而以甘露醇作为唯一碳源时,发酵产物中乙醇浓度为0.389g/L,NADH/NAD+比值和乙醇/乙酸的比值分别为1.04和16.0。     

Kinetic analysis of insulin action on amino acid uptake by isolated chick embryo heart cells

1. Isolated chick embryo heart cells were used to investigate the mode of action of insulin on the transport of three naturally occurring amino acids: l-proline, l-serine and glycine. Initial velocities of uptake were measured over a period of 5min with an 80-fold range of amino acid concentration. Corrections for amino acid diffusion, incorporation into protein and conversion into carbon dioxide were introduced. 2. The uptake processes approximated Michaelis-Menten kinetics within definite ranges of amino acid concentrations. A single transport system for proline and at least two transport systems for serine and glycine were detected. 3. The kinetic effects of insulin on transport systems for the amino acids tested were consistent with an acceleration of the maximal velocity of the process, without substantial changes in substrate concentration for half-maximal transport velocity. 4. These hormonal effects were not essentially altered by the corrections for amino acid incorporation into protein and conversion into carbon dioxide.