Physiological, biochemical, and mathematical studies of micro-aerobic continuous ethanol fermentation by Saccharomyces cerevisiae. I: hysteresis, oscillations, and maximum specific ethanol productivities in chemostat culture

The growth and metabolism of Saccharomyces cerevisiae was studied in steady-state chemostat cultures under conditions of scarce oxygen and excess glucose. The specific ethanol productivity and specific glucose uptake rate were stimulated by 50% within a narrow range of air/nitrogen mixtures to the fermentor. Fermentation was inhibited at slightly higher and lower air/nitrogen ratios, confirming similar results by previous investigators. This stimulation could not be caused by obvious mechanisms, such as the Pasteur or Crabtree effects. Since this maximum in the fermentation rate occurred in a steady-state chemostat and at a constant dilution rate, the ATP yield of the culture necessarily attained a minimum. Thus, changes in the energetic efficiency of growth or the degree of wasting of ATP were surmised. The steady-state biomass concentration at various oxygenation rates exhibited hysteresis phenomena. Ignition and extinction of the biomass concentration occurred as critical oxygen feed rates were passed. The hysteresis was prevented by adding yeast extract to or reducing the antifoam concentration in the medium. These medium alterations had the simultaneous effect of stimulating the fermentation rate, suggesting that ATP has a critical role in dictating the biomass concentration in micro-aerobic culture. Silicone polymer antifoam was found to stimulate glycerol production at the expense of ethanol production, having consequences for the energy generation and the biomass concentration of the culture.     

Measurement and analysis of intracellular ATP levels in metabolically engineered Zymomonas mobilis fermenting glucose and xylose mixtures

Intracellular adenosine-5'-triphosphate (ATP) levels were measured in a metabolically engineered Zymomonas mobilis over the course of batch fermentations of glucose and xylose mixtures. Fermentations were conducted over a range of pH (5-6) in the presence of varying initial amounts of acetic acid (0-8 g/L) using a 10% (w/v) total sugar concentration (glucose only, xylose only, or 5% glucose/5% xylose mixture). Over the design space investigated, ethanol process yields varied between 56.6% and 92.3% +/- 1.3% of theoretical, depending upon the test conditions. The large variation in process yields reflects the strong effect pH plays in modulating the inhibitory effect of acetic acid on fermentation performance. A corresponding effect was observed on maximum cellular specific growth rates, with the rates varying between a low of 0.15 h(-1) observed at pH 5 in the presence of 8 g/L acetic acid to a high of 0.32 +/- 0.02 h(-1) obtained at pH 5 or 6 when no acetic acid was initially present. While substantial differences were observed in intracellular specific ATP concentration profiles depending upon fermentation conditions, maximum intracellular ATP accumulation levels varied within a relatively narrow range (1.5-3.8 mg ATP/g dry cell mass). Xylose fermentations produced and accumulated ATP at much slower rates than mixed sugar fermentations (5% glucose, 5% xylose), and the ATP production and accumulation rates in the mixed sugar fermentations were slightly slower than in glucose fermentations. Results demonstrate that higher levels of acetic acid delay the onset and influence the extent of intracellular ATP accumulation. ATP production and accumulation rates were most sensitive to acetic acid at lower values of pH.     

Intracellular ethanol accumulation in yeast cells during aerobic fermentation: a Raman spectroscopic exploration

Aims: To investigate the intracellular ethanol accumulation in yeast cells by using laser tweezers Raman spectroscopy (LTRS). Methods and Results: Ethanol accumulation in individual yeast cells during aerobic fermentation triggered by excess glucose was studied using LTRS. Its amount was obtained by comparing intracellular and extracellular ethanol concentrations during initial process of ethanol production. We found that (i) yeasts start to produce ethanol within 3 min after triggering aerobic fermentation, (ii) average ratio of intracellular to extracellular ethanol is 1·54 ± 0·17 during the initial 3 h after addition of 10% (w/v) excess glucose and (iii) the accumulated intracellular ethanol is released when aerobic fermentation is stimulated with decreasing glucose concentration. Conclusions: Intracellular ethanol accumulation occurs in initial stage of a rapid aerobic fermentation and high glucose concentration may attribute to this accumulation process. Significance and Impact of the Study: This work demonstrates LTRS is a real‐time, reagent‐free, in situ technique and a powerful tool to study kinetic process of ethanol fermentation. This work also provides further information on the intracellular ethanol accumulation in yeast cells.     

限磷对产甘油假丝酵母甘油合成与胞内磷积累的影响

研究了磷酸盐限量对产甘油假丝酵母甘油合成与胞内磷积累的影响。结果表明, 当酵母细胞从适磷或富磷培养基转接入低磷培养基时, 发酵过程中胞内积累的磷逐渐减少; 而当菌体从低磷培养基转接入适磷或富磷培养基时, 发酵过程中胞内聚磷酸盐的积累量迅速增加。当细胞在第14小时和第38小时从适磷培养基转接入低磷培养基时甘油得率分别高达60.9%和61.4%, 而甘油产率则分别为2.03 g/(L·h)和2.23 g/(L·h)。这些现象说明限制发酵培养基中的磷浓度是产甘油假丝酵母高产甘油的必要条件, 并为其反复分批发酵法生产甘油提供了重要依据。     

Effect of Oxygenation on Xylose Fermentation by Pichia stipitis

The effect of oxygen limitation on xylose fermentation by Pichia stipitis (CBS 6054) was investigated in continuous culture. The maximum specific ethanol productivity (0.20 g of ethanol g dry weight⁻¹ h⁻¹) and ethanol yield (0.48 g/g) was reached at an oxygen transfer rate below 1 mmol/liter per h. In the studied range of oxygenation, the xylose reductase (EC 1.1.1.21) and xylitol dehydrogenase (EC 1.1.1.9) activities were constant as well as the ratio between the NADPH and NADH activities of xylose reductase. No xylitol production was found. The pyruvate decarboxylase (EC 4.1.1.1) activity increased and the malate dehydrogenase (EC 1.1.1.37) activity decreased with decreasing oxygenation. With decreasing oxygenation, the intracellular intermediary metabolites sedoheptulose 7-phosphate, glucose 6-phosphate, fructose 1,6-diphosphate, and malate accumulated slightly while pyruvate decreased. The ratio of the xylose uptake rate under aerobic conditions, in contrast to that under anaerobic assay conditions, increased with increasing oxygenation in the culture. The results are discussed in relation to the energy level in the cell, the redox balance, and the mitochondrial function.     

Osmotic pressure effects and intracellular accumulation of ethanol in yeast during fermentation

Summary The intracellular accumulation of ethanol in yeast and its potential effects on growth and fermentation have been topics of controversy for the past several years. The determination of intracellular ethanol based on the exclusion of [¹⁴C]sorbitol to estimate aqueous cell volume was used to examine the question of intracellular ethanol accumulation. An intracellular accumulation of ethanol inSaccharomyces cerevisiae was observed during the early stages of fermentation. However, as fermentation continued, the intracellular and extracellular concentrations of ethanol became similar. Increasing the osmotic pressure of the medium with glucose or sorbitol was observed to cause an increase in the intracellular ethanol concentration. Associated with this was a decrease in yeast growth and fermentation rates. In addition, increasing the osmotic pressure of the medium was observed to cause an increase in glycerol production. Supplementation of the media with excess peptone, yeast extract, magnesium sulfate and potassium phosphate was found to relieve the detrimental effects of high osmotic pressure. Under these conditions, though, no effect on the intracellular and extracellular ethanol distribution was observed. These results indicate that nutrient limitation, and not necessarily intracellular ethanol accumulation, plays a key role during yeast fermentations in media of high osmolarity.     

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.     

废糟液全循环对自絮凝酵母糖酵解途径关键酶、胁迫相关代谢物及胞内组分的影响

旨在研究废糟液直接全循环对絮凝酵母乙醇发酵、糖酵解关键酶以及细胞组成的影响。在一有效容积1.5 L的搅拌式生物反应器中,使用葡萄糖为220 g/L,添加8 g/L酵母粉和6 g/L蛋白胨的培养基,以0.04 h?1的稀释率进行自絮凝颗粒酵母乙醇连续发酵。每隔3天将收集到的发酵液集中精馏处理,得到的废糟液用于配制发酵培养基。装置运行近20 d,实验结果表明,随着废液循环批次的增加,系统乙醇和生物量浓度明显降低,糖酵解途径3个关键限速酶:己糖激酶、6-磷酸果糖激酶和丙酮酸激酶不同程度受到抑制。为了应对废糟液中高沸点副产物积累导致的环境胁迫,维持细胞正常代谢,甘油和菌体胞内蛋白生物合成加强,碳水化合物积累减弱。这些研究结果对进一步研究高沸点副产物积累对酵母细胞乙醇发酵影响的机理和菌种的代谢工程改造,具有重要意义。     

Physiological, biochemical, and mathematical studies of micro-aerobic continuous ethanol fermentation by Saccharomyces cerevisiae. III: mathematical model of cellular energetics and catabolism

Mathematical models of the catabolic pathways, the utilization and waste of ATP, and the factors affecting yeast growth in a micro-aerobic chemostat are presented. The models incorporate the intracellular metabolite and enzyme activity assays performed in Part II to explain the unusual macroscopic chemostat behaviors reported in Part I. The catabolic model successfully predicts a maximum in the specific ethanol productivity as a function of the intracellular ATP concentration. The ATP balance model enables the prediction of the intracellular ATP concentration and the ATP yield for given dissolved oxygen concentrations. Finally, in the context of a growth model, singularity theory provides a framework to explain the transition observed in Part I between hysteresis and the monotonic biomass versus oxygenation profiles in response to changes in the nutrient composition. The models serve to organize data and to concretely express proposed metabolic mechanisms and cause-effect hypotheses. The model is only applicable to the micro-aerobic and excess glucose conditions encountered in this study.     

Kinetics and metabolism of Bifidobacterium adolescentis MB 239 growing on glucose, galactose, lactose, and galactooligosaccharides

The kinetics and the metabolism of Bifidobacterium adolescentis MB 239 growing on galactooligosaccharides (GOS), lactose, galactose, and glucose were investigated. An unstructured unsegregated model for growth in batch cultures was developed, and kinetic parameters were calculated with a recursive algorithm. The growth rate and cellular yield were highest on galactose, followed by lactose and GOS, and were lowest on glucose. Lactate, acetate, and ethanol yields allowed the calculation of carbon fluxes toward fermentation products. Distributions between two- and three-carbon products were similar on all the carbohydrates (55 and 45%, respectively), but ethanol yields were different on glucose, GOS, lactose, and galactose, in decreasing order of production. Based on the stoichiometry of the fructose-6-phosphate shunt and on the carbon distribution among the products, the ATP yield was calculated. The highest yield was obtained on galactose, while the yields were 5, 8, and 25% lower on lactose, GOS, and glucose, respectively. Therefore, a correspondence among ethanol production, low ATP yields, and low biomass production was established, demonstrating that carbohydrate preferences may result from different distributions of carbon fluxes through the fermentative pathway. During the fermentation of a GOS mixture, substrate selectivity based on the degree of polymerization was exhibited, since lactose and the trisaccharide were the first to be consumed, while a delay was observed until longer oligosaccharides were utilized. Throughout the growth on both lactose and GOS, galactose accumulated in the cultural broth, suggesting that beta(1-4) galactosides can be hydrolyzed before they are taken up.     

Kinetic study of a change in intracellular ATP level associated with aerobic catabolism of ethanol by Streptococcus mutans.

Streptococcus mutans, a group of lactic acid bacteria and a normal inhabitant of the human oral cavity, generates ATP by substrate-level phosphorylation coupled to oxidation of ethanol (an end product of fermentation of sugars) into acetate in the presence of oxygen (K. Fukui, K. Kato, Kodama, H. Ohta, T. Shima moto, and T. Shimono, Proc. Jpn. Acad. 64B:13-16, 1988). Kinetic measurements were made of the cellular responses of S. mutans FA-1 to ethanol in comparison with those to glucose. In contrast to oxygen-independent acid production from glucose, oxygen was absolutely required for acid production from ethanol. Ethanol elicited a marked increase in the intracellular ATP concentration (ATPi) from a starved level to a steady level which was held constant as long as oxygen was present in the medium. Once oxygen was exhausted, ATPi returned to the starved level without delay. On the contrary, ATPi changes induced by glucose, which were independent of oxygen, followed a rather complicated time course before a steady level was established. Both the steady ATPi and the rate of accompanying oxygen consumption were functions of the ethanol concentration. These two parameters were linearly correlated, indicating that the unimolecular ATP turnover rate, which is independent of the rate of ATP generation in the steady state, can be calculated for cells energized by ethanol. The estimated turnover rate was 1.5 s-1 at 37 degrees C, which is comparable to that for other bacteria energized by glucose under nongrowing conditions.     

Transient increase in Ca2+ influx in Saccharomyces cerevisiae in response to glucose: effects of intracellular acidification and cAMP levels

Influx of 45Ca2+ into Saccharomyces cerevisiae was measured under experimental conditions which enabled measurements of initial rate of transport across the plasma membrane, without interference by the vacuolar Ca2+ transport system. Addition of glucose or glycerol to the cells, after pre-incubation in glucose-free medium for 5 min, caused a rapid, transient increase in 45Ca2+ influx, reaching a peak at 3-5 min after addition of substrate. Ethanol, or glycerol added with antimycin A, had no effect on 45Ca2+ influx. We have shown previously that this increase is not mediated by an effect of the substrates on intracellular ATP levels. Changes in membrane potential accounted for only a part of the glucose-stimulated 45Ca2+ influx. The roles of intracellular acidification and changes in cellular cAMP in mediating the effects of glucose on 45Ca2+ influx were examined. After a short preincubation in glucose-free medium addition of glucose caused a decrease in the intracellular pH, [pH]i, which reached a minimum value after 3 min. A transient increase in the cellular cAMP level was also observed. Addition of glycerol also caused intracellular acidification, but ethanol or glycerol added with antimycin A had no effect on [pH]i. Artificial intracellular acidification induced by exposure to isobutyric acid or to CCCP caused a transient rise in Ca2+ influx but the extent of the increase was smaller than that caused by glucose, and the time-course was different. We conclude that intracellular acidification may be responsible for part of the glucose stimulation of Ca2+ influx.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics and Metabolism of Bifidobacterium adolescentis MB 239 Growing on Glucose, Galactose, Lactose, and Galactooligosaccharides

The kinetics and the metabolism of Bifidobacterium adolescentis MB 239 growing on galactooligosaccharides (GOS), lactose, galactose, and glucose were investigated. An unstructured unsegregated model for growth in batch cultures was developed, and kinetic parameters were calculated with a recursive algorithm. The growth rate and cellular yield were highest on galactose, followed by lactose and GOS, and were lowest on glucose. Lactate, acetate, and ethanol yields allowed the calculation of carbon fluxes toward fermentation products. Distributions between two- and three-carbon products were similar on all the carbohydrates (55 and 45%, respectively), but ethanol yields were different on glucose, GOS, lactose, and galactose, in decreasing order of production. Based on the stoichiometry of the fructose-6-phosphate shunt and on the carbon distribution among the products, the ATP yield was calculated. The highest yield was obtained on galactose, while the yields were 5, 8, and 25% lower on lactose, GOS, and glucose, respectively. Therefore, a correspondence among ethanol production, low ATP yields, and low biomass production was established, demonstrating that carbohydrate preferences may result from different distributions of carbon fluxes through the fermentative pathway. During the fermentation of a GOS mixture, substrate selectivity based on the degree of polymerization was exhibited, since lactose and the trisaccharide were the first to be consumed, while a delay was observed until longer oligosaccharides were utilized. Throughout the growth on both lactose and GOS, galactose accumulated in the cultural broth, suggesting that β(1-4) galactosides can be hydrolyzed before they are taken up.     

粗糙脉孢菌(Neurospora crassa)木糖发酵的研究

研究了不同通氧条件和培养基初始pH等对粗糙脉孢菌(Neurospora crassa)AS3．1602木糖发酵的影响。结果表明,粗糙脉孢菌具有较强的发酵木糖产生乙醇及木糖醇的能力。通气量对木糖发酵有较大的影响。乙醇发酵适合在半好氧条件下进行,此时乙醇的转化率达到63．2％。木糖醇发酵适合在微好氧的条件下进行,转化率达到31．8％。木糖醇是在培养基中乙醇达到一定浓度后才开始积累。培养基的初始pH对木糖发酵产物有较大的影响,乙醇产生最适pH5．0,木糖醇产生最适pH4．0。在培养基pH为碱性条件时,木糖发酵受到很大的抑制。初始木糖浓度对产物乙醇及木糖醇的产率有很大的影响。葡萄糖的存在会抑制木糖的利用,对乙醇和木糖醇的产生也有很大的影响。     

Metabolic control analysis of glycerol synthesis in Saccharomyces cerevisiae

Glycerol, a major by-product of ethanol fermentation by Saccharomyces cerevisiae, is of significant importance to the wine, beer, and ethanol production industries. To gain a clearer understanding of and to quantify the extent to which parameters of the pathway affect glycerol flux in S. cerevisiae, a kinetic model of the glycerol synthesis pathway has been constructed. Kinetic parameters were collected from published values. Maximal enzyme activities and intracellular effector concentrations were determined experimentally. The model was validated by comparing experimental results on the rate of glycerol production to the rate calculated by the model. Values calculated by the model agreed well with those measured in independent experiments. The model also mimics the changes in the rate of glycerol synthesis at different phases of growth. Metabolic control analysis values calculated by the model indicate that the NAD(+)-dependent glycerol 3-phosphate dehydrogenase-catalyzed reaction has a flux control coefficient (C(J)v1) of approximately 0.85 and exercises the majority of the control of flux through the pathway. Response coefficients of parameter metabolites indicate that flux through the pathway is most responsive to dihydroxyacetone phosphate concentration (R(J)DHAP= 0.48 to 0.69), followed by ATP concentration (R(J)ATP = -0.21 to -0.50). Interestingly, the pathway responds weakly to NADH concentration (R(J)NADH = 0.03 to 0.08). The model indicates that the best strategy to increase flux through the pathway is not to increase enzyme activity, substrate concentration, or coenzyme concentration alone but to increase all of these parameters in conjunction with each other.     

Cofactor engineering in Saccharomyces cerevisiae: Expression of a H2O-forming NADH oxidase and impact on redox metabolism

The pyridine nucleotides NAD(H) and NADP(H) play major roles in the formation of by-products. To analyse how Saccharomyces cerevisiae (S. cerevisiae) metabolism during growth on glucose might be altered when intracellular NADH pool is decreased, we expressed noxE encoding a water-forming NADH oxidase from Lactococcus lactis (L. lactis) in the S. cerevisiae strain V5. During batch fermentation under controlled microaeration conditions, expression of the NADH oxidase under the control of a yeast promoter lead to large decreases in the intracellular NADH concentration (five-fold) and NADH/NAD+ ratio (six-fold). This increased NADH consumption caused a large redistribution of metabolic fluxes. The ethanol, glycerol, succinate and hydroxyglutarate yields were significantly reduced as a result of the lower NADH availability, whereas the formation of more oxidized metabolites, acetaldehyde, acetate and acetoin was favoured. The biomass yield was low and consumption of glucose, at concentration above 10%, was impaired. The metabolic redistribution in cells expressing the NADH oxidase was a consequence of the maintenance of a redox balance and of the management of acetaldehyde formation, which accumulated at toxic levels early in the process.     

Osmotic effects on bacterial transport and energetics

Paracoccus denitrificans suspended in media containing 20-300 mM NaCl swelled progressively as the salt concentration was decreased. The increase in intracellular water volume was accompanied by an enhancement of respiration and a stimulation of the rates of net potassium and alpha-aminoisobutyric acid accumulation. It is postulated that influx of water and consequent lowering of intracellular solute concentration trigger transport mechanisms which are destined to restore the original ion and metabolite balance. Since a number of transport reactions operate against the electrochemical gradient of their substrates, energy utilization increases. The increased ATP usage and lowering of [ATP] stimulates the activity of the respiratory chain and increases oxygen uptake and energy production.     

Glucose Utilization and Ethanolic Fermentation by Wild Type and Extrachromosomal Mutants of Neurospora crassa

Logarithmic growth rates, maximal biomass, specific glucose utilization rates, and ethanol accumulation were measured in aerobic cultures of wild type and extrachromosomal mutants of Neurospora crassa. Maximal biomass and ethanol accumulation of wild type and [mi-1] were proportional to the initial glucose concentration in the range of 2 to 10%. The specific rates of glucose utilization by the mutants were 13- to 20-fold greater than those of wild type in young cultures. The specific rates of glucose utilization by wild type, however, were increased threefold by increasing the ammonium ion concentration in the preculture medium. The suppressor gene f(+) suppressed the excessive glucose utilization and enhanced the growth rate and maximal biomass of [mi-1]. When the mutants were utilizing glucose at excessive rates, ethanol did not appear in the culture medium. Ethanol accumulation was maximum at stationary phase or thereafter, but there was little difference between the maxima of the mutants and wild type. The molar efficiency of the conversion of glucose to ethanol during the entire culture period of wild type and mutants was about 50% and, in the latter stages of fermentation, approached 100%. Replacement of ammonium ion by nitrate in the culture medium suppressed ethanol accumulation by wild type. The relationship of these results to previous observations on respiratory adaptation are discussed. We suggest that the Pasteur effect, the inhibition of fermentation by respiration, may be operative in N. crassa. Factors such as nitrogen source and concentration and oxygen tension, which may serve primarily to regulate the amount and form of respiration would, therefore, indirectly regulate fermentation. The mutants, although transiently deficient in terminal respiratory activity, do not accumulate more ethanol than wild type and, therefore, apparently do not ferment in excess to obtain additional adenosine 5'-triphosphate. We suggest that the excess activity of the alternate form of respiration of the mutants may be related to their excessive rate of glucose utilization by way of the pentose phosphate pathway and the oxidation of excess reduced nicotinamide adenine dinucleotide.     

By-product inhibition effects on ethanolic fermentation by Saccharomyces cerevisiae

Inhibition by secondary fermentation products may limit the ultimate productivity of new glucose to ethanol fermentation processes. New processes are under development whereby ethanol is selectively removed from the fermenting broth to eliminate ethanol inhibition effects. These processes can concentrate minor secondary products to the point where they become toxic to the yeast. Vacuum fermentation selectively concentrates nonvolatile products in the fermentation broth. Membrane fermentation systems may concentrate large molecules which are sterically blocked from membrane transport. Extractive fermentation systems, employing nonpolar solvents, may concentrate small organic acids. By-product production rates and inhibition levels in continuous fermentation with Saccharomyces cerevisiae have been determined for acetaldehyde, glycerol, formic, lactic, and acetic acids, 1-propanol, 2-methyl-1-butanol, and 2,3-butanediol to assess the potential effects of these by-products on new fermentation processes. Mechanisms are proposed for the various inhibition effects observed.