Continuous cultivation of dilute-acid hydrolysates to ethanol by immobilized Saccharomyces cerevisiae

The continuous cultivation of immobilized Saccharomyces cerevisiae CBS 8066 on dilute-acid hydrolysates of forest residuals was investigated. The yeast cells were immobilized in 2–4% Ca-alginate beads. The 2% beads were not stable. However, the 3 and 4% beads were stable for at least 3 wk when an extra resource of calcium ions was available in the medium. The continuous cultivation of a dilute-acid hydrolysate by the immobilized cells at dilution rates of 0.3, 0.5, and 0.6 h⁻¹ resulted in 86, 83, and 79% sugar consumption, respectively, and an ethanol yield between 0.45 and 0.48 g/g. The hydrolysate was fermentable at a dilution rate of 0.1 h⁻¹ in a free-cell system but washed out at a dilution rate of 0.2 h⁻¹. The continuous cultivation of a more inhibiting hydrolysate was not successful by either free- or immobilized-cell systems even at a low dilution rate of 0.07 h⁻¹. However, when the hydrolysate was overlimed, it was fermentable by the immobilized cells at a dilution rate of 0.2 h⁻¹.     

Ethanol-induced changes in glycolipids of Saccharomyces cerevisiae

Total glycolipid content of Saccharomyces cerevisiae cells increased in ethanol-treated yeast cells. Sialic acid and hexosamine contents of glycolipids from ethanol-treated cells decreased, whereas those of hexoses increased. Increased sialidase activity in the presence of ethanol may be responsible for the decrease in sialic acid content of glycolipids. The saccharide moieties of glycolipids of S. cerevisiae consisted of fucose, mannose, galactose, and glucose. Ethanol treatment of yeast cells caused an increase in glucose and a decrease in galactose content of glycolipids. The changes in glucose content can be related to changes in β-glucosidase activity under alcohol stress. The content of cerebrosides, sulfatides, and monoglucosyldiglycerides was enhanced following ethanol treatment. An increase in cerebroside as well as in sulfatide content during alcohol stress might play an important role in stabilizing the membrane both physically and structurally. Such variations in glycolipid content and composition of S. cerevisiae cells may represent an adaptive response to ethanol stress.     

Effect of agitation and aeration on production of hexokinase by Saccharomyces cerevisiae

A batch culture of Saccharomyces cerevisiae for the production of hexokinase was carried out in a 5-L fermentor containing 3 L of culture medium, which was in oculated with cell suspension (about 0.7 g/L), and left ferm entingat 35°C and pH 4.0. The aeration and agitation were adjusted to attain k _La values of 15, 60, 135, and 230 h⁻¹. The highest hexokinase productivity (754.6 U/[L h]) and substrate-cell conversion yield (0.21 g/g) occurred for a k _La of 60 h⁻¹. Moreover, the formation of hexokinase and cell growth are coupled events, which is in accordance with the constitutive character of this enzyme. Hexokinase formation for k _La>60 h⁻¹ was not enhanced probably owing to saturation of the respiratory pathway by oxygen.     

Production of ceramide with Saccharomyces cerevisiae

The possibility of producing the biologically active material of the skin, ceramide, was studied using yeasts. The yeast strain that produced the most ceramide, Saccharomyces cerevisiae (KCCM 50515), was selected, and the optimal conditions for ceramide production were determined using shakeflask culture and batch fermentation. By measuring the production rate of ceramide at various pH values and temperatures, the optimal conditions for ceramide production were found to be pH 6.0 and 30°C. When heat shock was applied to the cells for 1 h by increasing the culture temperature from 30 to 40°C after cell growth, the amount of ceramide produced was increased 5.9-fold. A cell growth and ceramide production model was developed with Monod kinetics and the Leudecking-Piret model. It showed that ceramide production was increased when the cells were in the stationary phase.     

Nitrogen regulation of Saccharomyces cerevisiae invertase

The regulation of extracellular enzymes is of great biotechnological interest. We studied the regulatory role of the URE 2 gene on the periplasmic invertase of Saccharomyces cerevisiae, because its periplasmic asparaginase is regulated by the URE2/GLN3 system. Enzymatic activity was measured in the isogenic strains P40-1B, the ure2 mutant P40-3C, and the P40-3C strain transformed with the pIC-CS plasmid carrying the URE2 gene. The assays were performed using midlog and stationary phase cells and nitrogen-starved cells from these growth phases. During exponential growth, the level of invertase in both wild-type and ure 2 mutant cells was comparable. However, the invertase activity in ure2 mutant cells from stationary phase was sixfold lower than in the wild-type cells. When P40-3C cells were transformed with the pIC-CS plasmid, the wild-type phenotype was restored. On nitrogen starvation in the presence of sucrose, the invertase activity in wild-type cells from midlog phase decreased three times, whereas in stationary cells, the activity decreased eight times. However, invertase activity doubled in ure 2 mutant cells from both phases. When these cells were trans-formed with the aforementioned plasmid, the wild-type phenotype was restored, although a significant invertase decrease in stationary cells was not observed. These results suggested that the URE2 protein plays a role in invertase activity.     

L-malic acid production by entrappedSaccharomyces cerevisiae into polyacrylamide gel beads

The yeastSaccharomyces cerevisiae was entrapped within polyacrylamide gel beads by employing a procedure that uses sodium dodecylsulfate as a detergent to improve the spherical configuration of the beads. The resulting preparation showed a rate of fumarate bioconversion to L-malic acid about 60 times higher than that found for the free cells. Almost all fumarate was converted in 30 min of incubation. The thermal stability of the immobilized cells did not significantly differ from the free cells. An optimal pH of 5.7 was found for the immobilized preparation and no succinic acid was detected as a by-product in the incubation mixture.     

Continuous potable alcohol production by immobilizedSaccharomyces cerevisiae on mineral kissiris

A biocatalyst prepared by the immobilization of Saccharomyces cerevisiae on the surface of the mineral kissiris was used in the present study for continuous potable-alcohol production. An ethanol productivity (calculated on the basis of liquid volume) of 10.5 g/L/h was obtained at a 0.7/h dilution rate, 121 g/L sucrose content, and 29.6% conversion employing molasse as feed material. Glucose, raisin extracts, and molasse were successively used as feed materials without stopping the operation of the reactor for 6 mo. The ethanol productivity and yield remained constant during the operational-stability study of the reactor, carried out for 44 d. Biomass productivity, yield, and free-cell concentration in glucose, raisin extracts, and molasse were examined. Finally, a system with two continuous reactors joined successively was also studied in the present investigation.     

Overexpression of glucose-6-phosphate dehydrogenase in genetically modified Saccharomyces cerevisiae

Glucose-6-phosphate dehydrogenase (G6PD) (EC 1.1.1.49) is an abun dant enzyme in Saccharomyces cerevisiae. This enzyme is of great interest as an analytical reagent because it is used in a large number of quantitative assays. A strain of S. cerevisiae was genetically modified to improve G6PD production during aerobic culture. The modifications are based on cloning the G6PD sequence under the control of promoters that are upregulated by the carbon source used for yeast growth. The results showed that S. cerevisiae acquired from a commercial source and the same strain produced by aerobic cultivation under controlled conditions provided very similar G6PD. However, G6PD production by genetically modified S. cerevisiae produced very high enzyme activity and showed to be the most effective procedure to obtain glucose-6-phosphate dehydrogenase. As a consequence, the cost of producing G6PD can be significantly reduced by using strains that contain levels of G6PD up to 14-fold higher than the level of G6PD found in commercially available strains.     

Salt-induced changes in lipid composition and ethanol tolerance inSaccharomyces cerevisiae

The effect of salt stress on lipid composition and its relationship with ethanol tolerance inSaccharomyces cerevisiae was studied. Amounts of phospholipids as well as that of sterols decreased, whereas that of protein and glycolipids increased with increasing salt concentration. Relative proportion of amino phospholipids (phosphatidylethanolamine and phosphatidylserine) decreased, whereas that of phosphatidylcholine showed a reverse trend. Cells grown under increasing salt concentration were more resistant to ethanol-induced leakage of UV-absorbing substances, an index of ethanol endurance. Results showed an overlap between osmotolerance and ethanol tolerance in this strain.     

Effects of potassium on the ethanol production rate of Saccharomyces cerevisiae carrying the plasmid pCYG4 related with ammonia assimilation

The influence of potassium on ethanol production bySaccharomyces cerevisiae wild type and AR5 cells carrying the plasmid pCYG4 was investigated. This plasmid carries the glutamate dehydrogenase gene conferring an 11-fold higher level of expressed enzyme activity over the wild type cells. All experiments were carried out in batch culture with medium supplemented to different potassium concentrations up to 180 mM. Maximum ethanol production rate was observed in the AR5 cells grown in medium supplemented with 3.5 mM of potassium ions. Glucose uptake rate increased with increasing potassium up to 60 mM, but higher concentrations depressed glucose uptake rate in both strains. Furthermore, the wild type cells showed higher growth rate, ethanol production, and glucose consumption rate than the AR5 cells. These lower rates in the AR5 cells could be explained by repression of potassium uptake by an enhancement of ammonium feeding, and greater energy requirements by these cells due the presence of the plasmid.     

Fermentation process optimization of recombinant Saccharomyces cerevisiae for the production of human interferon-α2a

The effects of different culture conditions on the expression level of human interferon-α2a (IFN-α2a) by using recombinant yeast were investigated in a 2.6-Ljar fermentor. Appropriate supplement of glucose and the maintenance of residual glucose at a low level resulted in the reduction of ethanol formation and enhancement of the bioactivity of IFN-α2a to 4.9×10⁶ from 3.1×10⁶ IU/mL. When adenine was added evenlly for 10–20 h of fermentation into the basal culture medium at a speed of 2 μg/mL of medium/h, OD₆₀₀ was greatly increased to 24, and the protein increased to 276 mg/L. The content of ethanol generated was also reduced tremendously during the process and as a result, 1.3×10⁷ IU/mL of biologic activity was achieved. In the expression phase, pH had an important impact on expression level, which should be controlled at 5.5     

Kinetics of asparaginase II fermentation in Saccharomyces cerevisiae ure2dal80 mutant

Although the quality of nitrogen source affects fermentation product formation, it has been managed empirically, to a large extent, in industrial scale. Laboratory-scale experiments successfully use the high-cost proline as a nonrepressive source. We evaluated urea as a substitute for proline in Saccharomyces cerevisiae ure2dal80 fermentations for asparaginase II production as a model system for nitrogen-regulated external enzymes. Maximum asparaginase II levels of 265 IU/L were observed in early stationary-phase cells grown on either proline or urea, whereas in ammonium cells, the maximum enzyme level was 157 IU/L. In all cases, enzyme stability was higher in buffered cultures with an initial pH of 6.5.     

L-malic acid production using immobilized saccharomyces cerevisiae

L-Malate was produced from fumarate by using immobilized Saccharomyces cerevisiae cells entrapped in polyacrylamide. This preparation performed better when pretreated with malonate. Under the experimental conditions described here, succinate was not detected as a by-product of the reaction, as had been reported for other microorganisms.     

Fermentation kinetics of ethanol production from glucose and xylose by recombinant Saccharomyces 1400(pLNH33)

Fermentation kinetics of ethanol production from glucose, xylose, and their mixtures using a recombinant Saccharomyces 1400 (pLNH33) are reported. Single-substrate kinetics indicate that the specific growth rate of the yeast and the specific ethanol productivity on glucose as the substrate was greater than on xylose as a substrate. Ethanol yields from glucose and xylose fermentation were typically 95 and 80% of the theoretical yield, respectively. The effect of ethanol inhibition is more pronounced for xylose fermentation than for glucose fermentation. Studies on glucose-xylose mixtures indicate that the recombinant yeast co-ferments glucose and xylose. Fermentation of a 52.8 g/L glucose and 56.3 g/L xylose mixture gave an ethanol concentration of 47.9 g/L after 36 h. Based on a theoretical yield of 0.51 g ethanol/g sugars, the ethanol yield from this experiment (for data up to 24 h) was calculated to be 0.46 g ethanol/g sugar or 90% of the theoretical yield. The specific growth rate of the yeast on glucose-xylose mixtures was found to lie between the specific growth rate on glucose and the specific growth rate on xylose. Kinetic studies were used to develop a fermentation model incorporating the effects of substrate inhibition, product inhibition, and inoculum size. Good agreements were obtained between model predictions and experimental data from batch fermentation of glucose, xylose, and their mixtures.     

The influence of products and by-products obtained by drinking water electrolysis on microorganisms

It is the aim of this work to underline the chemical character of disinfection processes and to present the results of treatment experiments for artificially and electrochemically added chemicals focusing on chlorine components and hydrogen peroxide obtained on IrO₂/RuO₂ anodes. A discontinuous cell with rotating anode placed 4 mm above an IrO₂ expanded mesh cathode of same diameter was used for the generation of electrolysis products. The thermostated experiments were carried out at a temperature of 20 °C and in constant current mode. Artificial solutions prepared using sodium salts and deionised water were electrolysed. Samples were analysed using the DPD (N, N-diethyl-p-phenylendiamine) method. Escherichia coli K-12, Bacillus subtilis DSM 2277 and Saccharomyces cerevisiae Kolin were used as test-microorganisms. Morphological changes were studied using transmission electron microscopy. The results show that electrolysed water had a higher lethal efficiency than Ca(OCl)₂ of the same measured active chlorine concentration. During the killing period mainly inner cell parts of the microorganisms react chemically with the disinfectants. In sufficiently high concentrations and reaction times, the cell material continues to react. Cell walls or membranes can break-through as electron microscopy pictures show. Electrolysed water had a higher lethal efficiency on microorganisms. This behaviour demonstrates the existence of additional by-products.     

Effect of a nonmetabolizable analog of fructose-1,6-bisphosphate on glycolysis and ethanol production in strains ofSaccharomyces cerevisiae andEscherichia coli

Fructose-1,6-bisphosphate (F-1,6-P2) is an allosteric activator of two key enzymes of glycolysis: phosphofructokinase and pyruvate kinase. Regulation of glycolysis in a wild-typeSaccharomyces cerevisiae and a recombinantEscherichia coli by a dead-end structural analog of F-1,6-P2 was studied. 2,5-Anhydromannitol (2,5-AM), a structural analog of β-d-fructose, was used. On being taken up by the cells, 2,5-AM was converted into its monophosphate and diphosphate by the enzymes of the glycolytic pathway. The final product, 2,5-anhydromannitol-1,6-bisphosphate, could not be metabolized further and, therefore, accumulated inside the cells. Glucose and fructose were used as substrates. It was found that 2,5-AM at concentrations of 1 mM or less did not have any effect on either substrate consumption or ethanol production. At concentrations of 2,5-AM of 2.5 mM or greater, significant inhibition of both glucose and fructose was observed, with fructose inhibition much more severe. We discuss the possible mechanisms of glycolysis inhibition by 2,5-AM at high concentrations and the regulation of glycolysis by this compound.     

New alcohol resistant strains ofSaccharomyces cerevisiae species for potable alcohol production using molasse

Two alcohol resistant strains of Saccharomyces cerevisiae species were isolated from a Greek vineyard plantation. The strain AXAZ-1 gave a concentration of 17.6% v/v alcohol and AXAZ-2 16.5%, when musts from raisin and sultana grapes, respectively, were employed in alcoholic fermentations. They were found to be more alcohol tolerant and fermentative in the fermentation of molasse than the traditional baker's yeast. Specifically, using an initial [symbol: see text] Be density of 16 [symbol: see text] Be at the repeated batch fermentation process, in the first as well as fourth batch, the better AXAZ-1 gave final [symbol: see text] Be densities of 6.0 and 10.5 respectively, and the baker's yeast 11.6 and 14.5.     

Characterization of an immobilized yeast cells fluidized-bed bioreactor for ethanol fermentation

The operational characterization of a fluidized-bed bioreactor for ethanol fermentation using Ca-alginate immobilized yeast cells is described. An additional air stream is supplied to the fermenter to ensure and maintain satisfactory fluidization behavior of beads and to avoid slug formation. The influence of physical properties such as bead density and liquid density on the fluidization quality and stability are discussed.     

Gold nanorod-catalyzed luminol chemiluminescence and its selective determination of glutathione in the cell extracts of Saccharomyces cerevisiae

In this study, gold nanorods were firstly found to exhibit a tremendously higher catalytic activity towards luminol chemiluminescence (CL) than spherical gold nanoparticles. More importantly, ultra-trace aminothiols can cause a great CL decrease in the gold nanorod-catalyzed luminol system by the formation of Au-S covalent bonds on the ends of gold nanorods. Aminothiols can occupy the active sites of gold nanorods, and further interrupt the generation of the active oxygen intermediates. Other biomolecules including 19 standard amino acids, alcohols, organic acids and saccharides have no effect on gold nanorod-catalyzed luminol CL signals. Moreover, in order to evaluate the applicability and reliability of the proposed method, it was applied to the determination of glutathione in the cell extracts of Saccharomyces cerevisiae. Good agreements were obtained for the determination of glutathione in the cell extracts of S. cerevisiae between the present approach and a standard Alloxan method. The recoveries of glutathione were found to fall in the range between 96 and 105%. The calibration curve for glutathione was found to be linear from 0.05 to 100 nM, and the detection limit (S/N = 3) was 0.01 nM. The relative standard deviation (RSD) for five repeated measurements of 5.0 nM glutathione was 2.1%.