Die Hemmung der lichtinduzierten Chlorophyll-, Carotinoid- und Anthocyansynthese durch Äthanol

The Inhibition of the Light Induced Chlorophyll, Carotenoid and Anthocyan Synthesis by Ethanol. It is shown that already low concentrations of ethanol inhibit the light induced formation of pigments (chlorophylls, carotenoids, anthocyanins) in Raphanus-seedlings. The degree of inhibition depends on the ethanol concentration and rises with increasing incubation period. The inhibition of pigment synthesis is reversible, and immediately ceases when ethanol is removed. The formation of chlorophylls and anthocyanin is more repressed than that of carotenoids, and the formation of β-carotene to a higher extent than that of xanthophylls. It is concluded that ethanol inhibits pigment formation by inhibition of protein synthesis. These results indicate that ethanol and other lower alcohols cannot be used for the solubilizing of organic compounds when these are applied to plant tissues.     

Kinetic modeling of Candida shehatae ATCC 22984 on xylose and glucose for ethanol production

Candida shehatae ATCC 22984, a xylose-fermenting yeast, showed an ability to produce ethanol in both glucose and xylose medium. Maximum ethanol produced by the yeast was 48.8?g/L in xylose and 52.6?g/L in glucose medium with ethanol yields that varied between 0.3 and 0.4?g/g depended on initial sugar concentrations. Xylitol was a coproduct of ethanol production using xylose as substrate, and glycerol was detected in both glucose and xylose media. Kinetic model equations indicated that growth, substrate consumption, and product formation of C. shehatae were governed by substrate limitation and inhibition by ethanol. The model suggested that cell growth was totally inhibited at 40?g/L of ethanol and ethanol production capacity of the yeast was 52?g/L, which were in good agreement with experimental results. The developed model could be used to explain C. shehatae fermentation in glucose and xylose media from 20 to 170?g/L sugar concentrations.     

The effects of potassium and chloride ions on the ethanolic fermentation of sucrose by Zymomonas mobilis 2716

Summary The inclusion of specific salts in Zymomonas mobilis batch sucrose fermentations can limit by-product formation. Sorbitol and fructo-oligosaccharide formation can be reduced and ethanol production enhanced by manipulating mineral salt concentrations. Chloride salts reduced the production of biomass and sorbitol in favour of fructo-oligosaccharide formation at concentrations lower than 10 g NaCl/l or MgCl₂. Higher concentrations led to the accumulation of glucose and fructose. Low concentrations of KH₂PO₄ (<20 g/l) enhanced biomass formation, and the concomitant reduction in sorbitol and fructo-oligosaccharides favoured enhanced ethanol formation. At concentrations above 20 g/l, its effects were similar to those obtained with the chloride salts. Invertase addition at the start of fermentation increased sorbitol formation, whereas addition after the completion of sucrose hydrolysis resulted in the conversion of fructo-oligosaccharides formed into fructose or ethanol. Fermentation with 250 g/l of sugar-cane syrup ( = 130 g sucrose/l) in the presence of 8 g KH₂PO₄/l, with 0.05 g invertase/l added on the completion of sucrose hydrolysis, resulted in a conversion efficiency of 94% with complete carbon accountability, and only 7 g sorbitol/l. Offprint requests to: H. W. Doelle     

Rapid ethanol fermentation of cellulose hydrolysate. II. Product and substrate inhibition and optimization of fermentor design

High concentrations of both ethanol and sugar in the fermentation broth inhibit the growth of yeast cells and the rate of product formation. Inhibitory effects of ethanol on the yeast strain Saccharomyces cerevisiae NRRL-Y-132 were studied in batch and continuous chemostat cultures. Growth was limited by either glucose or ethanol. Feed medium was supplemented with different ethanol concentrations. Ethanol was found to inhibit growth and the activity of yeast to produce ethanol in a noncompetitive manner. A linear kinetic pattern for growth and product formation was observed according to μ = μ_m (1 – P/P_m) and v = v_m (1 – P/P_m′), where μ_m is the maximum specific growth rate at P = 0 (hr^?1); P_m is the maximum specific product formation rate at P = 0 (hr^?1); P_m is the maximum ethanol concentration above which cells do not grow (g/liter); P_m′ is the maximum ethanol concentration above which cells do not produce ethanol (g/liter). Substrate inhibition studies were carried out using short-time experimental techniques under aerobic and anaerobic condition. The degree of substrate inhibition was found to be higher than that has been reported for ethanol fermentation of pure sugar. The kinetic relationships thus obtained were used to compute growth, substrate utilization, and alcohol production patterns and have been discussed with reference to batch and continuous fermentation of enzymatically produced bagasse hydrolysate.     

Continuous ethanol production at high glucose concentrations by a passively immobilized Zymomonas mobilis system

Summary The effect of high glucose concentrations on continuous ethanol production by passively immobilized Zymomonas mobilis cells has been studied. High effluent ethanol concentrations always led to low productivities. The maximum ethanol concentration attained was 92.8 g/l (98% glucose conversion) at a dilution rate of 0.14 h^-1 with 200 g/l glucose medium. The observed enhancement of cell immobilization in the fibrous support at high glucose concentrations in the feed input seems to be related to the formation of bacterial filaments.Preliminary results from this work were previously presented at the Second Spanish Conference on Biotechnology (Barcelona, 1988)     

Continuous measurement of ethanol production by aerobic yeast suspensions with an enzyme electrode

Summary An alcohol electrode was constructed which consisted of an oxygen probe onto which alcohol oxidase was immobilized. This enzyme electrode was used, in combination with a reference oxygen electrode, to study the short-term kinetics of alcoholic fermentation by aerobic yeast suspensions after pulsing with glucose. The results demonstrate that this device is an excellent tool in obtaining quantitative data on the short-term expression of the Crabtree effect in yeasts.Samples from aerobic glucose-limited chemostat cultures of Saccharomyces cerevisiae not producing ethanol, immediately (within 2 min) exhibited aerobic alcoholic fermentation after being pulsed with excess glucose. With chemostat-grown Candida utilis, however, ethanol production was not detectable even at high sugar concentrations. The Crabtree effect in S. cerevisiae was studied in more detail with commercial baker's yeast. Ethanol formation occurred only at initial glucose concentrations exceeding 150 mg·l^-1, and the rate of alcoholic fermentation increased with increasing glucose concentrations up to 1,000 mg·l^-1 glucose.Similar experiments with batch cultures of certain non-fermentative yeasts revealed that these organisms are capable of alcoholic fermentation. Thus, even under fully aerobic conditions, Hansenula nonfermentans and Candida buffonii produced ethanol after being pulsed with glucose. In C. buffonii ethanol formation was already apparent at very low glucose concentrations (10 mg·l^-1) and alcoholic fermentation even proceeded at a higher rate than in S. cerevisiae. With Rhodotorula rubra, however, the rate of ethanol formation was below the detection limit, i.e., less than 0.1 mmol·g cells^-1·h^-1.     

A reduced specific ethanol-forming performance of yeast at high biomass concentrations as a result of a changed ethanol-tolerance behaviour of the cells under condition of limitation. II. Proving the effect in high-flow rate fermentations with saccharomyces cerevisiae Sc 5

In growth-factor limited fermentations carried out in continuous high-flow rate fermenters the relationship existing between the specific ethanol formation rate and the ethanol concentration was found to change its characteristics from a linear function into a nonlinear one at high biomass concentrations. The deviation from the linear inhibitory behaviour of the cells was the greater the more the biomass concentration increased. A very good correspondence between the experimentally found productivities of ethanol formation and its adequate values obtained by calculation could be attained by using an improved steady-state productivity model, in which the variability of the function v = f(P) had been considered.     

Ethanol disrupts the formation of hypochord and dorsal aorta during the development of embryonic zebrafish

Exposure to ethanol during human embryonic period has severe teratogenic effects on the cardiovascular system. In our study, we demonstrated that ethanol of gradient concentrations can interfere with the establishment of circulatory system in embryonic zebrafish. The effective concentration to cause 50% malformations (EC₅₀) was 182.5 mmol/L. The ethanol pulse exposure experiment displayed that dome stage during embryogenesis is the sensitive time window to ethanol. It is found that 400 mmol/L ethanol pulse exposure can induce circulatory defects in 43% treated embryos. We ruled out the possibility that ethanol can interfere with the process of hematopoiesis in zebrafish. By employing in situ hybridization with endothelial biomarker (Flk-1), we revealed that ethanol disrupts the establishment of trunk axial vasculature, but has no effect on cranial vessels. Combined with the results of semi-thin histological sections, the in situ hybridization experiments with arterial and venous biomarkers (ephrinB2, ephB4) suggested that ethanol mainly interrupts the development of dorsal aorta while has little effect on axial vein. Further study indicated the negative influence of ethanol on the development of hypochord in zebrafish. The consequent lack of vasculogenic factors including Radar and Ang- 1 partly explains the defects in formation and integrity of dorsal aorta. These results provide important clues to the study of adverse effects of ethanol on the cardiovascular development in human fetus.     

Aerobic and anaerobic ethanol production by<Emphasis Type="Italic"> Mucor circinelloides</Emphasis> during submerged growth

The dimorphic organism Mucor circinelloides is currently being investigated as a potential host for heterologous protein production. The production of ethanol on pentose and hexose sugars was studied in submerged batch cultivations to further the general knowledge of Mucor physiology, with a view to the minimisation or elimination of the by-product ethanol for future process design. Large amounts of ethanol were produced during aerobic growth on glucose under non-oxygen limiting conditions, which is indicative of M. circinelloides being a Crabtree-positive organism. Ethanol production on galactose or xylose was less significant. The response of the organism to increased ethanol concentrations, both as the sole carbon source and in the presence of a sugar, was investigated in terms of biomass formation and morphology.     

Rhizomorph Formation in Fungi III. The Effect of Ethanol on the Synthesis of DNA and RNA and Uptake of Asparagine and Phosphate in Armillaria mellea

Growth and rhizomorph formation in Armillaria mellea (Vahl ex Fr.) Quél. can be stimulated by ethanol when grown on a synthetic glucose medium. The content of DNA (deoxy-ribonucleic acid) and RNA (ribonucleic acid) in cultures of A. mellea have been followed during a growth period in relation to growth (dry weight) and ethanol uptake with 3 different initial ethanol concentrations. The continuous increase in dry weight as well as DNA and RNA contents during growth showed similar exponential rates as long as ethanol was present in the medium. The final amounts were proportional to the initial ethanol concentration. A further supply of ethanol caused a similar proportional increase in dry weight and also in the DNA and RNA contents, which indicates that the growth stimulated by ethanol is caused by cell divisions rather than an accumulation of lipids or polysaccharides. Uptake of asparagine and phosphate were also stimulated by ethanol.     

Electron microscopic investigations of zymomonas mobilis cells grown in low and high glucose concentrations

Summary Zymomonas mobilis cells grown in the presence of low and high glucose concentrations were examined by electron microscopy. Using ultrathin sectioning and agar diffusion method, significant changes in morphology were observed. Although the fine structure resembles that of a typical gram-negative bacterium, changes in glucose concentration and phases of growth lead to large cell wall vesicle or blebs formation. The possible implications of this morphological change to glucose uptake and ethanol formation are discussed.     

Fructose production by Zymomonas mobilis in fed-batch culture with minimal sorbitol formation

Summary Fed-batch cultures of Zymomonas mobilis (UQM 2864), a mutant unable to metabolise fructose, grown on diluted sugar cane syrup (200 g/l sucrose) achieved yields of 90.5 g/l fructose and 48.3 g/l ethanol with minimal sorbitol formation and complete utilization of the substrate. The effect of inoculum size on sorbitol formation in the batch stage of fed-batch fermentation are reported. Fermentation of sucrose (350 g/l) supplemented with nutrients yielded 142 g/l fructose and 76.5 g/l ethanol. Some fructose product loss at high fructose concentrations was observed. The fed-batch fermentation process offers a method for obtaining high concentrations of fructose and ethanol from sucrose materials.     

Production of exopolysaccharide from ethanol inAgrobacterium radiobacter

Agrobacterium radiobacter produces an extracellular polysaccharide from various carbon sources. The exopolysaccharide is produced from ethanol also in a minimal medium containing nitrate as the sole nitrogen source. On cultivation in a medium without CaCO₃ only a minute amount of ethanol is converted to the exopolysaccharide. Both ethanol and nitrate in higher concentrations inhibit the growth rate. In a medium with CaCO₃ the proportion of ethanol converted to the polysaccharide is about ten-fold higher and the inhibitory effect of ethanol and nitrate on the growth rate is analogous to that found in the medium without CaCO₃-Comparison of results of mass and electron balance with a kinetic model shows that the parameters obtained in cultivations with CaCO₃ are less reliable. The balances point to the possibility of formation of other extracellular products.     

Acetic acid tolerance in Drosophila is a prerequisite for ethanol tolerance

Acetic acid tolerance compared with ethanol tolerance of Drosophila simulans and six Drosophila melanogaster strains shows a curvilinear relation with apparent asymptotic hyperbolic profile. The upper limit of acetic acid tolerance is lower than that for ethanol. We compared strains which had pairwise identical alcohol dehydrogenase (ADH) coding regions but different genetic backgrounds. A positive regression existed for ethanol tolerance on ADH activity. Adh-null mutants with very low ethanol tolerances had appreciable acetic acid tolerances and as a consequence did not fit the curve. ADH-F and ADH-S strains selected for high ethanol tolerances had the ability to tolerate high ethanol concentrations even after selection had been relaxed for several years. These selected lines tolerated higher acetic acid concentrations than the non-selected original strains. We propose that intake of high concentrations of ethanol and oxidation into acetic acid induces esterification of ethanol and acetic acid into ethylacetate. This cannot take place after the intake of acetic acid only, which also gives a lower energy yield.     

Mechanochemical study of conformational transitions and melting of Li-, Na-, K-, and CsDNA fibers in ethanol–water solutions

Highly oriented fibers of Li-, Na-, K-, and CsDNA were prepared with a previously developed wet spinning method. The procedure gave a large number of equivalent fiber bundle samples (reference length, L₀, typically = 12–15 cm) for systematic measurements of the fiber length L in ethanol–water solutions, using a simple mechanochemical set up. The decrease in relative length L/L₀ with increasing ethanol concentration at room temperature gave evidence for the B-A transition centered at 76% (v/v) ethanol for NaDNA fibers and at 80 and 84% ethanol for K- and CsDNA fibers. A smaller decrease in L/L₀ of LiDNA fibers was attributed to the B-C transition centered at 80% ethanol. In a second type of experiment with DNA fibers in ethanol–water solutions, the heat-induced helix–coil transition, or melting, revealed itself in a marked contraction of the DNA fibers. The melting temperature T_m, decreased linearly with increasing ethanol concentration for fibers in the B-DNA ethanol concentration region. In the B-A transition region, Na- and KDNA fibers showed a local maximum in T_m. On further increase of the ethanol concentration, the A-DXA region followed with an even steeper linear decrease in T_m. The dependence on the identity of the counterion is discussed with reference to the model for groove binding of cations in B-DNA developed by Skuratovskii and co-workers and to the results from Raman studies of the interhelical bonds in A-DNA performed by Lindsay and co-workers. An attempt to apply the theory of Chogovadze and Frank-Kamenetskii on DNA melting in the B-A transition region to the curves failed. However, for Na- and KDNA the T_m dependence in and around the A-B transition region could be expressed as a weighted mean value of T_m of A- and B-DNA. On further increase of the ethanol concentration, above 84% ethanol for LiDNA and above about 90% ethanol for Na-, K-, and CsDNA, a drastic change occurred. T_m increased and a few percentages higher ethanol concentrations were found to stabilize the DNA fibers so that they did not melt at all, not even at the upper temperature limit of the experiments (～ 80°C). This is interpreted as being due to the strong aggregation induced by these high ethanol concentrations and to the formation of P-DNA. Many features of the results are compatible with the counterion–water affinity model. In another series of measurements, T_m of DNA fibers in 75% ethanol was measured at various salt concentrations. No salt effect was observed (with the exception of LiDNA at low salt concentrations). This result is supported by calculations within the Poisson–Boltzmann cylindrical cell model. © 1994 John Wiley & Sons, Inc.     

Rhizomorph Formation in Fungi

The effect on growth and rhizomorph formation of 12 alcohols (methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, iso-butyl alcohol, tert-butyl alcohol, 1-pentanol, iso-amyl alcohol, ethylene glycol and glycerol) at different concentrations has been examined for 2 isolates of Armillaria mellea (Vahl ex Fr.) Quél. and 1 of Clitocybe geotropa (Bull. ex Fr.) Quél. The fungi were cultivated for 28 days on a synthetic, liquid glucose medium with the alcohols as supplement. The following alcohols strongly stimulated growth and rhizomorph formation: ethanol, 1-propanol and 1-butanol. A great variation was demonstrated between the isolates in relation to rhizomorph production, morphology, and ability to be stimulated by different alcohols.     

Fermentation and metabolic characteristics of Gluconacetobacter oboediens for different carbon sources

The metabolism of Gluconacetobacter oboediens was investigated in relation to different carbon sources for the continuous cultures at the dilution rate of 0.05 h⁻¹. The ¹³C-flux result implies the formation of metabolic recycles for the case of using glucose and acetate as carbon sources. When glucose and ethanol were used as carbon sources, the specific ethanol uptake rate and the specific acetate production rate increased as the feed ethanol concentration was increased from 40 to 60 g/l, while the specific CO₂ production rate and the biomass concentration decreased, where the ¹³C-metabolic flux result indicates that the glycolysis, oxidative PP pathway, and the tricarboxylic acid (TCA) cycle were less active, resulting in less biomass concentration. The flux result also implies that oxaloacetate decarboxylase flux became negative, so that oxaloacetate is backed up by this pathway, resulting in less activity of glyoxylate pathway. When gluconate was added for the case of using glucose and ethanol as carbon sources, the acetate and cell concentrations as well as gluconate concentrations increased. The glucose and ethanol concentrations decreased concomitantly with the increased feed gluconate concentration. In accordance with these fermentation characteristics, the enzyme activity result indicates that glucose dehydrogenase and glucose-6-phosphate dehydrogenase pathways became less active, while the glycolysis and the TCA cycle was activated as the feed gluconate concentration was increased.     

Ethanol induced the formation of β‐sheet and amyloid‐like fibrils by surfactant‐like peptide A6K

Self‐assembly of natural or designed peptides into fibrillar structures based on β‐sheet conformation is a ubiquitous and important phenomenon. Recently, organic solvents have been reported to play inductive roles in the process of conformational change and fibrillization of some proteins and peptides. In this study, we report the change of secondary structure and self‐assembling behavior of the surfactant‐like peptide A6K at different ethanol concentrations in water. Circular dichroism indicated that ethanol could induce a gradual conformational change of A6K from unordered secondary structure to β‐sheet depending upon the ethanol concentration. Dynamic light scattering and atomic force microscopy revealed that with an increase of ethanol concentration the nanostructure formed by A6K was transformed from nanosphere/string‐of‐beads to long and smooth fibrils. Furthermore, Congo red staining/binding and thioflavin‐T binding experiments showed that with increased ethanol concentration, the fibrils formed by A6K exhibited stronger amyloid fibril features. These results reveal the ability of ethanol to promote β‐sheet conformation and fibrillization of the surfactant‐like peptide, a fact that may be useful for both designing self‐assembling peptide nanomaterials and clarifying the molecular mechanism behind the formation of amyloid fibrils. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Relationship Between Effect of Ethanol on Proton Flux Across Plasma Membrane and Ethanol Tolerance, inPichia stipitis

Pichia stipitisefficiently converts glucose or xylose into ethanol but is inhibited by ethanol concentrations exceeding 30 g/L. InSaccharomyces cerevisiae, ethanol has been shown to alter the movement of protons into and out of the cell. InP. stipitisthe passive entry of protons into either glucose- or xylose-grown cells is unaffected at physiological ethanol concentrations. In contrast, active proton extrusion is affected differentially by ethanol, depending on the carbon source catabolized. In fact, in glucose-grown cells, the H⁺-extrusion rate is reduced by low ethanol concentrations, whereas, in xylose-grown cells, the H⁺-extrusion rate is reduced only at non-physiological ethanol concentrations. Thus, the ethanol inhibitory effect on growth and ethanol production, in glucose-grown cells, is probably caused by a reduction in H⁺-extrusion. Comparison of the rates of H⁺-flux with the relatedin vitroH⁺-ATPase activity suggests a new mechanism for the regulation of the proton pumping plasma membrane ATPase (EC 3.6.1.3) ofP. stipitis, by both glucose and ethanol. Glucose activates both the ATP hydrolysis and the proton-pumping activities of the H⁺-ATPase, whereas ethanol causes an uncoupling between the ATP hydrolysis and the proton-pumping activities. This uncoupling may well be the cause of ethanol induced growth inhibition of glucose grownP. stipitiscells.