Acylphosphatase increases the rate of ethanol production from glucose in cell-free extracts of Saccharomyces cerevisiae

Addition of acylphosphatase exerted a stimulating effect on the alcoholic fermentation of glucose by Saccharomyces cerevisiae. The rates of glucose degradation and ethanol production by cell-free extracts of the S-288C strain were measured in the absence and in the presence of various levels of this enzyme. Two acylphosphatase isoenzymes were used; one was purified from horse skeletal muscle and the other from human erythrocytes. Both increased the rate of alcoholic fermentation, but that from erythrocytes proved to be the more efficient. This stimulating action is probably due to an "uncoupling effect" of acylphosphatase on the fermentative process, through hydrolysis of 3-phosphoglyceroyl phosphate. This was demonstrated by the fact that alcoholic fermentation was stimulated considerably by a mixture of ADP and inorganic phosphate and by arsenate as well. The possibility of improving the fermentative capacity of microorganisms may have important biotechnological applications.     

Glucose transport in crabtree-positive and crabtree-negative yeasts

The kinetic parameters of glucose transport in four Crabtree-positive and four Crabtree-negative yeasts were determined. The organisms were grown in aerobic glucose-limited chemostats at a dilution rate of 0.1 h-1. The results show a clear correlation between the presence of high-affinity glucose transport systems and the absence of aerobic fermentation upon addition of excess glucose to steady-state cultures. The presence of these H+-symport systems could be established by determination of intracellular accumulation of 6-deoxy-[3H]glucose and alkalinization of buffered cell suspensions upon addition of glucose. In contrast, the yeasts that did show aerobic alcoholic fermentation during these glucose pulse experiments had low-affinity facilitated-diffusion carriers only. In the yeasts examined the capacity of the glucose transport carriers was higher than the actual glucose consumption rates during the glucose pulse experiments. The relationship between the rate of sugar consumption and the rate of alcoholic fermentation was studied in detail with Saccharomyces cerevisiae. When S. cerevisiae was pulsed with low amounts of glucose or mannose, in order to obtain submaximal sugar consumption rates, fermentation was already occurring at sugar consumption rates just above those which were maintained in the glucose-limited steady-state culture. The results are interpreted in relation with the Crabtree effect. In Crabtree-positive yeasts, an increase in the external glucose concentration may lead to unrestricted glucose uptake by facilitated diffusion and hence, to aerobic fermentation. In contrast, Crabtree-negative yeasts may restrict the entry of glucose by their regulated H+-symport systems and thus prevent the occurrence of overflow metabolism.     

The role of peroxisomes in xylose alcoholic fermentation in the engineered Saccharomyces cerevisiae

Xylose is a second‐most abounded sugar after glucose in lignocellulosic hydrolysates and should be efficiently fermented for economically viable second‐generation ethanol production. Despite significant progress in metabolic and evolutionary engineering, xylose fermentation rate of recombinant Saccharomyces cerevisiae remains lower than that for glucose. Our recent study demonstrated that peroxisome‐deficient cells of yeast Ogataea polymorpha showed a decrease in ethanol production from xylose. In this work, we have studied the role of peroxisomes in xylose alcoholic fermentation in the engineered xylose‐utilizing strain of S. cerevisiae. It was shown that peroxisome‐less pex3Δ mutant possessed 1.5‐fold decrease of ethanol production from xylose. We hypothesized that peroxisomal catalase Cta1 may have importance for hydrogen peroxide, the important component of reactive oxygen species, detoxification during xylose alcoholic fermentation. It was clearly shown that CTA1 deletion impaired ethanol production from xylose. It was found that enhancing the peroxisome population by modulation the peroxisomal biogenesis by overexpression of PEX34 activates xylose alcoholic fermentation.     

葡萄酒酿酒酵母嗜果糖特性的评价方法

[目的]酿酒酵母的嗜果糖性是葡萄酒酵母选育工作的一项重要内容.建立评价菌体发酵果糖能力的方法,是葡萄酒酿酒酵母嗜果糖性研究的基础.[方法]以3株不同果糖发酵能力的酵母菌为研究对象,考察菌体在模拟葡萄汁培养基条件下,发酵情况与单糖利用之间的关系;并通过数学方程拟合单糖动力发酵曲线,得到发酵持续时间、葡萄糖浓度拟为0时的果糖浓度、果糖与葡萄糖曲线面积的差值等参数.[结果]这些参数可以反应出菌体的发酵速率和嗜果糖性.其中后两个参数能显著将3个菌株的嗜果糖特性区分开.[结论]为高果糖利周优良葡萄酒酿酒酵母菌株的筛选和构建,提供了较为全面、客观和有效的评价方法.     

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.     

Effects of ADH2 overexpression in Saccharomyces bayanus during alcoholic fermentation

The effect of overexpression of the gene ADH2 on metabolic and biological activity in Saccharomyces bayanus V5 during alcoholic fermentation has been evaluated. This gene is known to encode alcohol dehydrogenase II (ADH II). During the biological aging of sherry wines, where yeasts have to grow on ethanol owing to the absence of glucose, this isoenzyme plays a prominent role by converting the ethanol into acetaldehyde and producing NADH in the process. Overexpression of the gene ADH2 during alcoholic fermentation has no effect on the proteomic profile or the net production of some metabolites associated with glycolysis and alcoholic fermentation such as ethanol, acetaldehyde, and glycerol. However, it affects indirectly glucose and ammonium uptakes, cell growth, and intracellular redox potential, which lead to an altered metabolome. The increased contents in acetoin, acetic acid, and L-proline present in the fermentation medium under these conditions can be ascribed to detoxification by removal of excess acetaldehyde and the need to restore and maintain the intracellular redox potential balance.     

Localization and kinetics of pyruvate-metabolizing enzymes in relation to aerobic alcoholic fermentation in Saccharomyces cerevisiae CBS 8066 and Candida utilis CBS 621

The role of pyruvate metabolism in the triggering of aerobic, alcoholic fermentation in Saccharomyces cerevisiae has been studied. Since Candida utilis does not exhibit a Crabtree effect. this yeast was used as a reference organism. The localization, activity and kinetic properties of pyruvate carboxylase (EC 6.4.1.1), the pyruvate dehydrogenase complex and pyruvate decarboxylase (EC 4.1.1.1) in cells of glucose-limited chemostat cultures of the two yeasts were compared. In contrast to the general situation in fungi, plants and animals, pyruvate carboxylase was found to be a cytosolic enzyme in both yeasts. This implies that for anabolic processes, transport of C4-dicarboxylic acids into the mitochondria is required. Isolated mitochondria from both yeasts exhibited the same kinetics with respect to oxidation of malate. Also, the affinity of isolated mitochondria for pyruvate oxidation and the in situ activity of the pyruvate dehydrogenase complex was similar in both types of mitochondria. The activity of the cytosolic enzyme pyruvate decarboxylase in S. cerevisiae from glucose-limited chemostat cultures was 8-fold that in C. utilis. The enzyme was purified from both organisms, and its kinetic properties were determined. Pyruvate decarboxylase of both yeasts was competitively inhibited by inorganic phosphate. The enzyme of S. cerevisiae was more sensitive to this inhibitor than the enzyme of C. utilis. The in vivo role of phosphate inhibition of pyruvate decarboxylase upon transition of cells from glucose limitation to glucose excess and the associated triggering of alcoholic fermentation was investigated with 31P-NMR. In both yeasts this transition resulted in a rapid drop of the cytosolic inorganic phosphate concentration. It is concluded that the relief from phosphate inhibition does stimulate alcoholic fermentation, but it is not a prerequisite for pyruvate decarboxylase to become active in vivo. Rather, a high glycolytic flux and a high level of this enzyme are decisive for the occurrence of alcoholic fermentation after transfer of cells from glucose limitation to glucose excess.     

Metabolic engineering of the initial stages of xylose catabolism in yeasts for construction of efficient producers of ethanol from lignocelluloses

Plant biomass possesses a huge potential as a source for biofuel production. The main components of biomass are glucose and five-carbon sugar xylose. The yeast Saccharomyces cerevisiae that is used for industrial ethanol production from glucose is unable to xylose fermentation. Therefore a microorganism capable for efficient fermentation of both glucose and xylose has to be found in nature or constructed for economically feasible biomass conversion to ethanol. The active xylose fermentation could be performed by increasing the efficiency of initial stages of xylose metabolism. In this review the enzymes of initial stages of xylose metabolism in yeasts (xylose reductase, xylitol dehydrogenase, xylulokinase) and bacteria (xylose isomerase and xylulokinase) are characterized. The ways for construction of yeast strains capable of efficient alcoholic xylose fermentation are discussed.     

Fermentation effects of oligosaccharides of Radix Ophiopogonis on alloxan-induced diabetes in mice

In this study, oligosaccharides extracted from Ophiopogon japonicus vinegar (OOV) by alcoholic and acetic acid fermentation with water extracts from Radix Ophiopogon and oligosaccharides extracted from Radix Ophiopogonis (OOJ) were investigated. Characterization of the extracts indicated that OOV are proteoglycans, whereas OOJ are not. Moreover, compared with OOJ, monosaccharide compositions of OOV only include fructose and galactose and not glucose. MALDI-TOF-mass spectrometric results showed that the molecular weight of OOV was smaller after fermentation. Changes in the characteristics of OOV would inevitably lead to changes in its hypoglycemic properties. The OOV inhibition activity against α-glucosidase was stronger than that of OOJ. The inhibition activity became stronger with higher dosages of OOV. The hypoglycemic effect of OOV on alloxan-induced diabetic mice was stronger than that of OOJ. More important, the ability of OOV to reduce damage on islets in diabetic mice was stronger than that of OOJ. Overall, alcoholic and acetic acid fermentation improved the hypoglycemic activity of OOJ.     

Anoxia tolerance and anaerobic catabolism of aged beetroot storage tissues5

The relationship between anoxia tolerance and rates of anaerobiccatabolism was studied using aged storage tissues of red beetroot.This tissue has substantial experimental advantages over mostother tissues for studies on response to anoxia; even the aeratedtissues do not grow, thus allowing assesment of any possiblePasteur effect. Furthermore, loss of the endogeneous dye, betacyanin,serves as a marker for death of individual cells. A high tolerance to anoxia was achieved by adding glucose andby applying a hypoxic pretreatment for 48 h (02 at 0.003–0.015mol m^– Such tissues survived anoxia for at least 150 h. Alcoholic fermentation was the principal catabolic pathway inanoxic beetroot tissue; ethanol synthesized over 88.5 h of anoxiaaccounted for about 86% of the decrease In endogenous sugarcontent in hypoxically pretreated tissues. During the first24 h of anoxia, rates of alcoholic fermentation were stimulatedby high concentrations of endogenous glucose, supplying exogenousglucose and also by hypoxic pretreatment. In aerobically pretreatedtissues, alcoholic fermentation increased with time over thefirst 24 h of anoxia and these increases were correlated withincreases in activity of pyruvate decarboxylase (PDC). The mostrapid rates of glycolysis under anoxia were observed in thepresence of glucose, c. 50% above the rate in aerated tissues. When anoxia lasted longer than 24 h, the rate of alcoholic fermentationdeclined with time, in all treatments. Furthermore, decreasesin content of endogen ous substrates (sugars + starch), between0 and 88.5 h anoxia, indicated that, in hypoxically pretreatedtissues, glycolysis was 30% lower under anoxia than in air.These decreases in rate of alcoholic fermentation were not dueto injury, or decreases in activity of PDC. Reduced availabilityof endogenous sugar can not be excluded as a cause for the decreasein rate of glycolysis. However, we favour fine control, whichwould regulate glycolysis once requirements for ATP are reduced,after adaptation to anoxia has been completed. We also estimatethat maintenance requirement for ATP is 10–25 times lowerIn anoxic than in aerated tissues. Key words: Anoxia tolerance, alcoholic fermentation, maintenance requirement, storage tissues     

Studies on the energy metabolism during anaerobic fermentation of glucose by baker's yeast

Summary As a result of the intimate association of ADP phosphorylation with alcoholic fermentation, resulting in the synthesis of 2 mole ATP per mole glucose fermented, it may be calculated that a minimum of 672 µcal heat development may be expected for every mm³ CO₂ developed during alcoholic fermentation. When all ATP produced would be fully de-phosphorylated to ADP + Pi (e.g. by ATP-ase activity) a maximum heat development of 1200 µcal per mm³ CO₂ could be expected.Using the LKB-Flow-Microcalorimeter for measurement of heat development and at the same time the Warburg technique for measuring CO₂ development during anaerobic glucose fermentation of a baker's yeast suspension, the heat development per mm³ CO₂ produced was calculated over a fermentation period of 90 min.Maintenance of strict anaerobic conditions in the Flow-Microcalorimeter vessel was complicated by diffusion of traces of oxygenvia the Teflon transport lines, resulting in excessive heat development values, not representative for the alcoholic fermentation. This problem could be circumvented by removal of traces of oxygen by means of addition of the enzyme glucose-oxidase.Poisoning the respiratory enzyme system of the yeast by addition of KCN or azide, or using respiratory-deficient mutants of the yeast also resulted in heat development values, inherent with alcoholic fermentation.The values obtained were very close to the minimum of 672 µcal per mm³ CO₂, at least during the initial phases of fermentation, indicating that ADP regeneration from ATP, essential for maintaining the high fermentation rate, is not primarily the result of ATP-ase activity, but must be due to participation of ATP in energy-requiring synthetic reactions.     

CO₂ from alcoholic fermentation for continuous cultivation of Arthrospira (Spirulina) platensis in tubular photobioreactor using urea as nitrogen source

Carbon dioxide released from alcoholic fermentation accounts for 33% of the whole CO(2) involved in the use of ethanol as fuel derived from glucose. As Arthrospira platensis can uptake this greenhouse gas, this study evaluates the use of the CO(2) released from alcoholic fermentation for the production of Arthrospira platensis. For this purpose, this cyanobacterium was cultivated in continuous process using urea as nitrogen source, either using CO(2) from alcoholic fermentation, without any treatment, or using pure CO(2) from cylinder. The experiments were carried out at 120 μmol photons m(-2) s(-1) in tubular photobioreactor at different dilution rates (0.2 ≤ D ≤ 0.8 d(-1) ). Using CO(2) from alcoholic fermentation, maximum steady-state cell concentration (2661 ± 71 mg L(-1) ) was achieved at D = 0.2 d(-1) , whereas higher dilution rate (0.6 d(-1) ) was needed to maximize cell productivity (839 mg L(-1) d(-1) ). This value was 10% lower than the one obtained with pure CO(2) , and there was no significant difference in the biomass protein content. With D = 0.8 d(-1) , it was possible to obtain 56% ± 1.5% and 50% ± 1.2% of protein in the dry biomass, using pure CO(2) and CO(2) from alcoholic fermentation, respectively. These results demonstrate that the use of such cost free CO(2) from alcoholic fermentation as carbon source, associated with low cost nitrogen source, may be a promising way to reduce costs of continuous cultivation of photosynthetic microorganisms, contributing at the same time to mitigate the greenhouse effect.     

Fermentation of citrate by Lactobacillus plantarum in the presence of a yeast under acid conditions

Summary At pH 3.6, Lactobacillus plantarum is unable to grow on citrate or to ferment it in the absence of another carbon source such as glucose. In a defined medium containing glucose and citrate, with a higher concentration of the former than the latter, as in many fermented alcoholic beverages, L. plantarum will first ferment the sugar. The production of lactate from glucose degradation increases the acidity of the medium and inhibits the fermentation of citrate. In co-culture with Saccharomyces cerevisiae, part of the glucose is fermented by the yeast, partly avoiding the pH drop and the inhibition of citrate fermentation by L. plantarum. Fermentation was still possible at pH values around 3.0. Offprint requests to: C. Kennes     

Effect of ethanol addition on propionic acid consumption in an anaerobic bioreactor

Summary Experiments were carried out during the start up of an anaerobic bioreactor fed with synthetic medium based on glucose. Results showed different substrate fermentative pathways, mainly alcoholic fermentation and anaerobic oxidation. External ethanol additions produced a decrease in the levels of propionic acid. This method for stabilization of anaerobic reactors was more efficient than others reported in the literature.     

Measurement of oxygen solubility in fermentation media: a colorimetric method

Methods of measuring oxygen solubility in culture media are scarce, and those available are tedious to apply. A simple colorimetric assay was developed and applied to the analysis of oxygen solubility during alcoholic fermentation. The method was based on the consumption of oxygen by glucose oxidase activity and the production of the pink quinone of syringaldazine by coupled peroxidase activity. Color formation at 526 nm progressed through an optimum that was a linear function of the oxygen added to the assay. Sensitivity was maximized by operating at pH 7 and limiting the medium sample volume added. Each assay took 10-15 min to prepare and react. Reaction time was minimized by using abundant glucose and enzyme concentrations. Data obtained by the assay developed showed good agreement with published oxygen solubilities in water and selected media at various temperatures. Subsequent analyses of fermentation broths indicated falling sugar concentration to be primarily responsible for increases in oxygen solubility during fermentation. For example, during fermentations started with 230 g/L xylose or glucose, oxygen solubility could increase by 41% due to sugar consumption alone. This procedure can provide the solubility data needed to accurately calibrate in-line electronic probes for monitoring dissolved oxygen concentration during fermentation processes.     

Differential pattern of trehalose accumulation in wine yeast strains during the microvinification process

Trehalose accumulation in wine yeast strains growing under microvinification conditions was determined and compared to that obtained under laboratory conditions. Industrial strains accumulate 10-fold more trehalose than laboratory strains. Contrary to batch-culture growth, under microvinification conditions trehalose accumulation is not consequence of glucose exhaustion. Physiological relevance of trehalose during the process of wine making and their use for potential improvements of alcoholic fermentation are discussed.     

Use of Saccharomyces cerevisiae cells immobilized on orange peel as biocatalyst for alcoholic fermentation

A biocatalyst was prepared by immobilizing a commercial Saccharomyces cerevisiae strain (baker's yeast) on orange peel pieces for use in alcoholic fermentation and for fermented food applications. Cell immobilization was shown by electron microscopy and by the efficiency of the immobilized biocatalyst for alcoholic fermentation of various carbohydrate substrates (glucose, molasses, raisin extracts) and at various temperatures (30-15 degrees C). Fermentation times in all cases were low (5-15 h) and ethanol productivities were high (av. 150.6 g/ld) showing good operational stability of the biocatalyst and suitability for commercial applications. Reasonable amounts of volatile by-products were produced at all the temperatures studied, revealing potential application of the proposed biocatalyst in fermented food applications, to improve productivities and quality.