Influence of cell concentration,temperature, and press performance on flow characteristics and disintegration in the freeze-pressing of Saccharomyces cerevisiae with the X-press

The pressure required for initiation of flow when freeze-pressing with the X-press is related to the phase boundaries of water, particularly those between ice I and liquid even at temperatures around ?25°C and lower. Widening the orifice of the pressure chamber to diameters larger than 2.5 mm leads to lower pressures and less extensive cell disintegration. Pressing Saccharomyces cerevisiae slowly with the aid of a manual hydraulic jack at ?25°C produces a disintegration of 60–75% irrespective of cell concentration. Pressing at ?35°C shows no clear differences. Pressing more rapidly with the aid of a motor-driven hydraulic press produces a similar extent of disruption of diluted cell suspensions (5.4 mg/g) as slow pressing. However, freeze-pressing a paste of baker's yeast (270 mg/g) increases the degree of disintegration. Under these conditions the disintegration is further enhanced by a lower temperature, ?35°C, and by a high velocity of flow through the orifice, such that more than 95% of the S. cerevisiae is disrupted by one pressing at less than 2 × 10⁸ Pa. Mechanisms for flow through the X-press are suggested and discussed in relation to the phase diagram of water.     

Response to chilling of tomato seedlings and cells in suspension cultures

Tomato cell suspensions and seedlings (Lycopersicon esculentum) responded comparably when exposed to chilling temperatures (10 C or below). Seedling growth and cellular activities related to cell viability and culture growth (triphenyltetrazolium chloride reduction, fluoroscein diacetate uptake, and hydrolysis) were sharply diminished below 10 C. Arrhenius plots of the respiratory O₂ consumption by both seedlings and cell suspensions had a break at 10 C, as is characteristic for chilling-sensitive species. The acyl chains that were found in the phospholipids of both cell cultures and seedlings were similar. These results indicate the potential usefulness of plant suspension cultures for studies of chilling injury.     

Study of the interaction of Saccharomyces cerevisiae with glucose by particle microelectrophoresis

Saccharomyces cerevisiae NCYC 239 in the presence of glucose at temperatures under 303 K shows a time-dependent lowering of electrophoreric mobility $\text{υ}$. At temperatures above 303 K, this time-dependent change in $\text{υ}$ is in the direction of increased mobilities. Cells suspended in buffer indicate a surface pK_a of less than 4, whereas for cells suspended in buffered glucose it is impossible to derive a surface pK_a. A kinetic study of the interaction of S. cerevisiae with glucose as a function of temperature allows calculation of an activation energy of 140 kJ·mol^?1 for the combined processes of (i) uptake of glucose onto the cell wall, (ii) transfer through the cell wall and membrane, and (iii) the establishment of a steady glucose flux through the wall and membrane.     

Fermentation Temperature Modulates Phosphatidylethanolamine and Phosphatidylinositol Levels in the Cell Membrane of Saccharomyces cerevisiae

During alcoholic fermentation, Saccharomyces cerevisiae is exposed to a host of environmental and physiological stresses. Extremes of fermentation temperature have previously been demonstrated to induce fermentation arrest under growth conditions that would otherwise result in complete sugar utilization at “normal” temperatures and nutrient levels. Fermentations were carried out at 15°C, 25°C, and 35°C in a defined high-sugar medium using three Saccharomyces cerevisiae strains with diverse fermentation characteristics. The lipid composition of these strains was analyzed at two fermentation stages, when ethanol levels were low early in stationary phase and in late stationary phase at high ethanol concentrations. Several lipids exhibited dramatic differences in membrane concentration in a temperature-dependent manner. Principal component analysis (PCA) was used as a tool to elucidate correlations between specific lipid species and fermentation temperature for each yeast strain. Fermentations carried out at 35°C exhibited very high concentrations of several phosphatidylinositol species, whereas at 15°C these yeast strains exhibited higher levels of phosphatidylethanolamine and phosphatidylcholine species with medium-chain fatty acids. Furthermore, membrane concentrations of ergosterol were highest in the yeast strain that experienced stuck fermentations at all three temperatures. Fluorescence anisotropy measurements of yeast cell membrane fluidity during fermentation were carried out using the lipophilic fluorophore diphenylhexatriene. These measurements demonstrate that the changes in the lipid composition of these yeast strains across the range of fermentation temperatures used in this study did not significantly affect cell membrane fluidity. However, the results from this study indicate that fermenting S. cerevisiae modulates its membrane lipid composition in a temperature-dependent manner.     

Predicting the stabilities of freeze-dried suspensions of Lactobacillus acidophilus by the accelerated storage test

The elevated temperatures of 50 ° C, 60 ° C, and 70 ° C were used in an accelerated storage test for predicting the stability of freeze-dried suspensions of L. acidophilus. The logarithmic death of bacteria at the above temperatures and the Arrhenius relationship obtained permitted predicting the rate of death at any storage temperature. The values predicted for storage stability of freeze-dried suspensions of L. acidophilus at 4 ° C and 20 ° C were confirmed by the actual values obtained after storage at these temperatures for 6, 15, and 19 mo.     

Water permeability of yeast cells at sub-zero temperatures

Summary A combined cryomicroscopic-multiple nonlinear regression analysis technique has been used to determine the water permeability of the yeast cellSaccharomyces cerevisiae during freezing. The time rate of change in volume of supercooled yeast cells was photographically monitored using a cryomicroscope which is capable of controlling in a programmable manner both the temperature and the time rate of change in temperature of the cell suspension being studied. Multiple nonlinear regression analysis together with a thermodynamic model of cell water transport during freezing was then used to statistically deduce the subzero temperature dependence of the cell water permeability. The water permeability process forS. cerevisiae being cooled at subzero temperatures was found to be rate-limited by the passage of water through either the plasmalemma, the cell wall, or a combination of these two permeability barriers. The hydraulic water permeability coefficient for yeast at 20°C is approximately 1–2×10^–13 cm³/dyne sec, if extrapolation from subzero temperatures to room temperature is permissible, while the apparent activation energy governing the permeability process at subzero temperatures is approximately 45–68 kJ/mol (11–16 kcal/mol). Appendix I: Volumetric Changes in Yeast Cells during Freezing at Constant Cooling Rates     

Saccharomyces cerevisiae and Oenococcus oeni immobilized in different layers of a cellulose/starch gel composite for simultaneous alcoholic and malolactic wine fermentations

The production of a two-layer composite biocatalyst for immobilization of two different microorganisms for simultaneous alcoholic and malolactic fermentation (MLF) of wine in the same bioreactor is reported. The biocatalyst consisted of a tubular delignified cellulosic material (DCM) with entrapped Oenococcus oeni cells, covered with starch gel containing the alcohol resistant and cryotolerant strain Saccharomyces cerevisiae AXAZ-1. The biocatalyst was found effective for simultaneous low temperature alcoholic fermentation resulting to conversion of malic acid to lactic acid in 5 days at 10 °C. Improvement of wine quality compared with wine fermented with S. cerevisiae AXAZ-1 immobilized on DCM was attributed to MLF as well as to increased ester formation and lower higher alcohols produced at low fermentation temperatures (10 °C) as shown by GC and headspace SPME GC/MS analysis. Scanning electron microscopy showed that the preparation of a three-layer composite biocatalyst is also possible. The significance of such composite biocatalysts is the feasibility of two or three bioprocesses in the same bioreactor, thus reducing production cost in the food industry     

Photooxidative production of carbon monoxide by phototrophic microorganisms

Net photosynthesis and CO production were measured in cell suspensions of Chlorella fusca. The rate of net photosynthesis showed saturation curves with increasing radiation intensities and CO₂-mixing ratios. Maximum rates were found at 35° C with a sharp decrease at higher temperatures. By contrast, the rate of CO production was proportional to the radiation intensity and did not show any saturation up to 1.5 kW · m^?2 white light. The CO-production rate was higher in blue than in red light and was independent of the CO₂-mixing ratio of the carrier gas within a range of 0–1000 ppmv. We found that the CO-production rate was constant within the physiological temperature range of 10–35° C, but increased considerably at higher temperatures and that CO production by the chlorophyll-deficient mutant of C. fusca was 5 times that of the wild type. In addition, we measured CO production in cell suspensions of Chromatium vinosum, Rhodopseudomonas sphaeroides and Rhodopseudomonas acidophila, which were grown either anaerobically in the light or aerobically in the dark. CO production could only be observed when the cells were incubated in the presence of oxygen and light. Under these conditions more CO was produced by aerobically grown cells than by phototrophically grown cells of R. sphaeroides and R. acidophila. The results obtained indicate that CO was produced by photosensitized oxidations and not by metabolic processes.     

Hybridization within Saccharomyces Genus Results in Homoeostasis and Phenotypic Novelty in Winemaking Conditions

Despite its biotechnological interest, hybridization, which can result in hybrid vigor, has not commonly been studied or exploited in the yeast genus. From a diallel design including 55 intra- and interspecific hybrids between Saccharomyces cerevisiae and S. uvarum grown at two temperatures in enological conditions, we analyzed as many as 35 fermentation traits with original statistical and modeling tools. We first showed that, depending on the types of trait – kinetics parameters, life-history traits, enological parameters and aromas –, the sources of variation (strain, temperature and strain * temperature effects) differed in a large extent. Then we compared globally three groups of hybrids and their parents at two growth temperatures: intraspecific hybrids S. cerevisiae * S. cerevisiae, intraspecific hybrids S. uvarum * S. uvarum and interspecific hybrids S. cerevisiae * S. uvarum. We found that hybridization could generate multi-trait phenotypes with improved oenological performances and better homeostasis with respect to temperature. These results could explain why interspecific hybridization is so common in natural and domesticated yeast, and open the way to applications for wine-making.     

High temperature stimulates acetic acid accumulation and enhances the growth inhibition and ethanol production by Saccharomyces cerevisiae under fermenting conditions

Cellular responses of Saccharomyces cerevisiae to high temperatures of up to 42 °C during ethanol fermentation at a high glucose concentration (i.e., 100 g/L) were investigated. Increased temperature correlated with stimulated glucose uptake to produce not only the thermal protectant glycerol but also ethanol and acetic acid. Carbon flux into the tricarboxylic acid (TCA) cycle correlated positively with cultivation temperature. These results indicate that the increased demand for energy (in the form of ATP), most likely caused by multiple stressors, including heat, acetic acid, and ethanol, was matched by both the fermentation and respiration pathways. Notably, acetic acid production was substantially stimulated compared to that of other metabolites during growth at increased temperature. The acetic acid produced in addition to ethanol seemed to subsequently result in adverse effects, leading to increased production of reactive oxygen species. This, in turn, appeared to cause the specific growth rate, and glucose uptake rate reduced leading to a decrease of the specific ethanol production rate far before glucose depletion. These results suggest that adverse effects from heat, acetic acid, ethanol, and oxidative stressors are synergistic, resulting in a decrease of the specific growth rate and ethanol production rate and, hence, are major determinants of cell stability and ethanol fermentation performance of S. cerevisiae at high temperatures. The results are discussed in the context of possible applications.     

Growth of the plankton diatom Thalassiosira nordenskioeldii Cleve at low silicate concentrations

The combined effect of low silicate concentration and temperature on the growth of the marine plankton diatom Thalassiosira nordenskioeldii Cleve was investigated by means of batch and semi-continuous cultures. Growth rates were measured in thin cell suspensions (less than 500 cells/ml) to prevent the silicate concentration in the medium from decreasing by more than 25 % during the period of measurement.Half-saturation constants of silicate-limited growth were calculated according to the Michaelis-Menten equation. At 3 °C, the constant was 0.09 μg-at. Si/I and at 10 °C, 0.02μg-at. Si/I. In semicontinuous cultures grown for one month at silicate concentrations of 0.3-0.4μg-at. Si/I, the mean cell division rates were 80–100% of the maximum rates recorded at the respective temperatures.It seems unlikely that decreasing silicate concentrations could influence the course of the spring succession of plankton diatom species in arctic or temperate coastal waters.     

Summer temperature can predict the distribution of wild yeast populations

The wine yeast, Saccharomyces cerevisiae, is the best understood microbial eukaryote at the molecular and cellular level, yet its natural geographic distribution is unknown. Here we report the results of a field survey for S. cerevisiae,S. paradoxus and other budding yeast on oak trees in Europe. We show that yeast species differ in their geographic distributions, and investigated which ecological variables can predict the isolation rate of S. paradoxus, the most abundant species. We find a positive association between trunk girth and S. paradoxus abundance suggesting that older trees harbor more yeast. S. paradoxus isolation frequency is also associated with summer temperature, showing highest isolation rates at intermediate temperatures. Using our statistical model, we estimated a range of summer temperatures at which we expect high S. paradoxus isolation rates, and show that the geographic distribution predicted by this optimum temperature range is consistent with the worldwide distribution of sites where S. paradoxus has been isolated. Using laboratory estimates of optimal growth temperatures for S. cerevisiae relative to S. paradoxus, we also estimated an optimum range of summer temperatures for S. cerevisiae. The geographic distribution of these optimum temperatures is consistent with the locations where wild S. cerevisiae have been reported, and can explain why only human‐associated S. cerevisiae strains are isolated at northernmost latitudes. Our results provide a starting point for targeted isolation of S. cerevisiae from natural habitats, which could lead to a better understanding of climate associations and natural history in this important model microbe.     

Genetic improvement of xylose metabolism by enhancing the expression of pentose phosphate pathway genes in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> IR-2 for high-temperature ethanol production

The pentose phosphate pathway (PPP) plays an important role in the efficiency of xylose fermentation during cellulosic ethanol production. In simultaneous saccharification and co-fermentation (SSCF), the optimal temperature for cellulase hydrolysis of lignocellulose is much higher than that of fermentation. Successful use of SSCF requires optimization of the expression of PPP genes at elevated temperatures. This study examined the combinatorial expression of PPP genes at high temperature. The results revealed that over-expression of TAL1 and TKL1 in Saccharomyces cerevisiae (S. cerevisiae) at 30 °C and over-expression of all PPP genes at 36 °C resulted in the highest ethanol productivities. Furthermore, combinatorial over-expression of PPP genes derived from S. cerevisiae and a thermostable yeast Kluyveromyces marxianus allowed the strain to ferment xylose with ethanol productivity of 0.51 g/L/h, even at 38 °C. These results clearly demonstrate that xylose metabolism can be improved by the utilization of appropriate combinations of thermostable PPP genes in high-temperature production of ethanol.     

The contuation of the phase-boundary between ice I and liquid into the region of ice III and II and its relation to freeze-pressing of biological material.

The trajectory of the phase-boundary between ice I and liquid has been continuously followed by compression of deionized water, 0.10 m KCl, 0.10 m NaCl, and deionized water with suspended yeast cells (Saccharomyces cerevisiae, 180 mg/g) in a close-ended pressure chamber at temperatures below 0 °. Upon increasing pressure on deionized H₂O at ?8.6 °C the temperature first increases, until the transition line between ice I and liquid is reached. Then the sample cools on further compression, which is concomitant with an increase in electrical conductivity, indicating the gradual formation of liquid. At ?34.8 °C the pressure drops spontaneously from 3 × 10⁸ to 2.4 × 10⁸ Pa, the conductivity decreases, and the volume of the samples becomes further reduced to ?3.1 cm³/mole of H₂O, making the formation of ice III probable. On increase of pressure on 0.10 m KCl and 0.10 m NaCl the sample is gradually cooled, as the fusion line of the respective eutectic solid is reached. 0.10 m KCl is then super-cooled into the region of ice III and II, whereas 0.10 m NaCl is desalinated with a final conductivity of the suspension of 3–10 nmho/cm. In the sample with S. cerevisiae 180 mg/g the ice I-liquid phase-boundary was followed to ?36.0 °C into the region and ice III and II.These results are of great importance to the understanding of the freeze-pressing process, since they indicate that a transition from ice I to liquid may occur even at temperatures between ?22 °C and ?35 °C, thus facilitating flow of material through the press. This way they shed light on the pressures needed to initiate flow at different temperatures and compositions of the sample to be freeze-pressed.     

Effect of Growth Conditions and Trehalose Content on Cryotolerance of Bakers' Yeast in Frozen Doughs

The cryotolerance in frozen doughs and in water suspensions of bakers' yeast (Saccharomyces cerevisiae) previously grown under various industrial conditions was evaluated on a laboratory scale. Fed-batch cultures were very superior to batch cultures, and strong aeration enhanced cryoresistance in both cases for freezing rates of 1 to 56°C min⁻¹. Loss of cell viability in frozen dough or water was related to the duration of the dissolved-oxygen deficit during fed-batch growth. Strongly aerobic fed-batch cultures grown at a reduced average specific rate (μ = 0.088 h⁻¹ compared with 0.117 h⁻¹) also showed greater trehalose synthesis and improved frozen-dough stability. Insufficient aeration (dissolved-oxygen deficit) and lower growth temperature (20°C instead of 30°C) decreased both fed-batch-grown yeast cryoresistance and trehalose content. Although trehalose had a cryoprotective effect in S. cerevisiae, its effect was neutralized by even a momentary lack of excess dissolved oxygen in the fed-batch growth medium.     

Regulation of Cell Division in a Temperature-Sensitive Division Mutant of Escherichia coli

Escherichia coli fil ts forms multinucleate filaments when suspensions of about 10(7) organisms per ml are shifted from 37 to 43 C in rich medium. Occasional septation continues, chiefly at the poles, and immediately becomes more frequent when the filaments are returned to 37 C. The addition of chloramphenicol (200 mug/ml) at either temperature initially stimulates the formation of polar septa. When very dilute suspensions of the strain (<10(6) organisms per ml) are shifted to the restrictive temperature, the inhibition of septation is more complete and only seldom reversible. Conversely, cell division is little affected when suspensions of >10(8) organisms per ml, or microcolonies of several hundred organisms on agar, are incubated at 43 C; evidence is presented that this is a consequence of a slight reduction in the mutant's growth rate. In certain media, septation is blocked irreversibly by even brief exposure to 43 C, after which cell elongation without division proceeds at 37 C for some hours. Several findings, when considered together, suggest that the cytoplasmic membrane is normal at the restrictive temperature, and that the block in septation is caused by a defect in the cell wall: it is largely overcome by NaCl, but not by sucrose; in some circumstances the filaments become swollen and develop localized bulges in the wall, yet the membrane remains intact and retains its selective permeability; lastly, the strain is insensitive to deoxycholate at both temperatures. The mutation has been mapped between arg B and thr, at a locus which appears to be distinct from others known primarily to influence cell division.     

Microbial Cells as Biosorbents for Heavy Metals: Accumulation of Uranium by Saccharomyces cerevisiae and Pseudomonas aeruginosa

Uranium accumulated extracellularly on the surfaces of Saccharomyces cerevisiae cells. The rate and extent of accumulation were subject to environmental parameters, such as pH, temperature, and interference by certain anions and cations. Uranium accumulation by Pseudomonas aeruginosa occurred intracellularly and was extremely rapid (<10 s), and no response to environmental parameters could be detected. Metabolism was not required for metal uptake by either organism. Cell-bound uranium reached a concentration of 10 to 15% of the dry cell weight, but only 32% of the S. cerevisiae cells and 44% of the P. aeruginosa cells within a given population possessed visible uranium deposits when examined by electron microscopy. Rates of uranium uptake by S. cerevisiae were increased by chemical pretreatment of the cells. Uranium could be removed chemically from S. cerevisiae cells, and the cells could then be reused as a biosorbent.     

Aerobic Mineralization of Trichloroethylene, Vinyl Chloride, and Aromatic Compounds by Rhodococcus Species

Two Rhodococcus strains which were isolated from a trichloroethylene (TCE)-degrading bacterial mixture and Rhodococcus rhodochrous ATCC 21197 mineralized vinyl chloride (VC) and TCE. Greater than 99.9% of a 1-mg/liter concentration of VC was degraded by cell suspensions. [1,2-¹⁴C]VC was degraded by cell suspensions, with the production of greater than 66% ¹⁴CO₂ and 20% ¹⁴C-aqueous phase products and incorporation of 10% of the ¹⁴C into the biomass. Cultures that utilized propane as a substrate were able to mineralize greater than 28% of [1,2-¹⁴C]TCE to ¹⁴CO₂, with approximately 40% appearing in ¹⁴C-aqueous phase products and another 10% of ¹⁴C incorporated into the biomass. VC degradation was oxygen dependent and occurred at a pH range of 5 to 10 and temperatures of 4 to 35°C. Cell suspensions degraded up to 5 mg of TCE per liter and up to 40 mg of VC per liter. Propane competitively inhibited TCE degradation. Resting cell suspensions also degraded other chlorinated aliphatic hydrocarbons, such as chloroform, 1,1-dichloroethylene, and 1,1,1-trichloroethane. The isolates degraded a mixture of aromatic and chlorinated aliphatic solvents and utilized benzene, toluene, sodium benzoate, naphthalene, biphenyl, and n-alkanes ranging in size from propane to hexadecane as carbon and energy sources. The environmental isolates appeared more catabolically versatile than R. rhodochrous ATCC 21197. The data report that environmental isolates of Rhodococcus species and R. rhodochrous ATCC 21197 have the potential to degrade TCE and VC in addition to a variety of aromatic and chlorinated aliphatic compounds either individually or in mixtures.     

Synergistic Actions of Nisin, Sublethal Ultrahigh Pressure, and Reduced Temperature on Bacteria and Yeast

Nisin in combination with ultrahigh-pressure treatment (UHP) showed strong synergistic effects against Lactobacillus plantarum and Escherichia coli at reduced temperatures (<15°C). The strongest inactivation effects were observed when nisin was present during pressure treatment and in the recovery medium. Elimination (>6-log reductions) of L. plantarum was achieved at 10°C with synergistic combinations of 0.5 μg of nisin per ml at 150 MPa and 0.1 μg of nisin per ml at 200 MPa for 10 min. Additive effects of nisin and UHP accounted for only 1.2- and 3.7-log reductions, respectively. Elimination was also achieved for E. coli at 10°C with nisin present at 2 μg/ml, and 10 min of pressure at 200 MPa, whereas the additive effect accounted for only 2.6-log reductions. Slight effects were observed even against the yeast Saccharomyces cerevisiae with nisin present at 5 μg/ml and with 200 MPa of pressure. Combining nisin, UHP, and lowered temperature may allow considerable reduction in time and/or pressure of UHP treatments. Kill can be complete without the frequently encountered survival tails in UHP processing. The slightly enhanced synergistic kill with UHP at reduced temperatures was also observed for other antimicrobials, the synthetic peptides MB21 and histatin 5. The postulated mode of action was that the reduced temperature and the binding of peptides to the membrane increased the efficacy of UHP treatment. The increases in fatty acid saturation or diphosphatidylglycerol content and the lysylphosphatidyl content of the cytoplasm membrane of L. plantarum were correlated with increased susceptibility to UHP and nisin, respectively.     

Simultaneous Saccharification and Ethanol Fermentation of Corn Stover at High Temperature and High Solids Loading by a Thermotolerant Strain Saccharomyces cerevisiae DQ1

The thermotolerant strain Saccharomyces cerevisiae DQ1 was applied to the simultaneous saccharification and fermentation (SSF) at high temperature and high solids loading of the dilute acid-pretreated corn stover in the present study. The SSF using S. cerevisiae DQ1 was operated at 30?% solids loading of the pretreated corn stover with three-step SSF mode and achieved up to ethanol titer of 48?g/L and yield of 65.6?%. S. cerevisiae DQ1 showed strong thermotolerance in both the regular one-step SSF and the three-step SSF with changing temperature in each step. The three-step SSF at 40°C using S. cerevisiae DQ1 tolerated the greater cellulase dosage and solids loading of the pretreated corn stover and resulted in increased ethanol production. The present study provided a practical potential for the future SSF of lignocellulose feedstock at high temperature to reach high ethanol titer.