Oxygen requirements of the food spoilage yeast Zygosaccharomyces bailii in synthetic and complex media

Most yeast species can ferment sugars to ethanol, but only a few can grow in the complete absence of oxygen. Oxygen availability might, therefore, be a key parameter in spoilage of food caused by fermentative yeasts. In this study, the oxygen requirement and regulation of alcoholic fermentation were studied in batch cultures of the spoilage yeast Zygosaccharomyces bailii at a constant pH, pH 3.0. In aerobic, glucose-grown cultures, Z. bailii exhibited aerobic alcoholic fermentation similar to that of Saccharomyces cerevisiae and other Crabtree-positive yeasts. In anaerobic fermentor cultures grown on a synthetic medium supplemented with glucose, Tween 80, and ergosterol, S. cerevisiae exhibited rapid exponential growth. Growth of Z. bailii under these conditions was extremely slow and linear. These linear growth kinetics indicate that cell proliferation of Z. bailii in the anaerobic fermentors was limited by a constant, low rate of oxygen leakage into the system. Similar results were obtained with the facultatively fermentative yeast Candida utilis. When the same experimental setup was used for anaerobic cultivation, in complex YPD medium, Z. bailii exhibited exponential growth and vigorous fermentation, indicating that a nutritional requirement for anaerobic growth was met by complex-medium components. Our results demonstrate that restriction of oxygen entry into foods and beverages, which are rich in nutrients, is not a promising strategy for preventing growth and gas formation by Z. bailii. In contrast to the growth of Z. bailii, anaerobic growth of S. cerevisiae on complex YPD medium was much slower than growth in synthetic medium, which probably reflected the superior tolerance of the former yeast to organic acids at low pH.     

Raman Spectroscopy and Chemometrics for Identification and Strain Discrimination of the Wine Spoilage Yeasts Saccharomyces cerevisiae,Zygosaccharomyces bailii,and Brettanomyces bruxellensis

The yeasts Zygosaccharomyces bailii, Dekkera bruxellensis (anamorph, Brettanomyces bruxellensis), and Saccharomyces cerevisiae are the major spoilage agents of finished wine. A novel method using Raman spectroscopy in combination with a chemometric classification tool has been developed for the identification of these yeast species and for strain discrimination of these yeasts. Raman spectra were collected for six strains of each of the yeasts Z. bailii, B. bruxellensis, and S. cerevisiae. The yeasts were classified with high sensitivity at the species level: 93.8% for Z. bailii, 92.3% for B. bruxellensis, and 98.6% for S. cerevisiae. Furthermore, we have demonstrated that it is possible to discriminate between strains of these species. These yeasts were classified at the strain level with an overall accuracy of 81.8%.     

Yeast Community Structures and Dynamics in Healthy and Botrytis-Affected Grape Must Fermentations

Indigenous yeast population dynamics during the fermentation of healthy and Botrytis-affected grape juice samples from two regions in Greece, Attica and Arcadia, were surveyed. Species diversity was evaluated by using restriction fragment length polymorphism and sequence analyses of the 5.8S internal transcribed spacer and the D1/D2 ribosomal DNA (rDNA) regions of cultivable yeasts. Community-level profiles were also obtained by direct analysis of fermenting samples through denaturing gradient gel electrophoresis of 26S rDNA amplicons. Both approaches revealed structural divergences in yeast communities between samples of different sanitary states or geographical origins. In all cases, Botrytis infection severely perturbed the bioprocess of fermentation by dramatically altering species heterogeneity and succession during the time course. At the beginning and middle of fermentations, Botrytis-affected samples possessed higher levels of biodiversity than their healthy counterparts, being enriched with fermentative and/or spoilage species, such as Zygosaccharomyces bailii and Issatchenkia spp. or Kluyveromyces dobzhanskii and Kazachstania sp. populations that have not been reported before for wine fermentations. Importantly, Botrytis-affected samples exposed discrete final species dominance. Selection was not species specific, and two different populations, i.e., Saccharomyces cerevisiae in samples from Arcadia and Z. bailii in samples from Attica, could be recovered at the end of Botrytis-affected fermentations. The governing of wine fermentations by Z. bailii is reported for the first time and could elucidate the origins and role of this particular spoilage microbe for the wine industry. This is the first survey to compare healthy and Botrytis-affected spontaneous fermentations by using both culture-based and -independent molecular methods in an attempt to further illuminate the complex yeast ecology of grape must fermentations.     

Effects of single and combined cell treatments based on low pH and high concentrations of ethanol on the growth and fermentation of Dekkera bruxellensis and Saccharomyces cerevisiae

The alcoholic fermentation in Brazil displays some peculiarities because the yeast used is recycled in a non-aseptic process. After centrifugation, the cells are treated with acid to control the bacterial growth. However, it is difficult to manage the indigenous yeasts without affecting the main culture of Saccharomyces cerevisiae. This work evaluated how the cell treatment could be modified to combat contaminant yeasts based on the differential sensitivities to low pH and high concentrations of ethanol displayed by an industrial strain of S. cerevisiae and three strains of Dekkera bruxellensis, which are common contaminant yeasts in Brazilian fermentation processes. The tests were initially performed in rich medium with a low pH or a high concentration of ethanol to analyse the yeast growth profile. Then, the single and combined effects of low pH and ethanol concentration on the yeast cell viability were evaluated under non-proliferative conditions. The effects on the fermentation parameters were also verified. S. cerevisiae grew best when not subjected to the stresses, but this yeast and D. bruxellensis had similar growth kinetics when exposed to a low pH or increased ethanol concentrations. However, the combined treatments of low pH (2.0) and ethanol (11 or 13 %) resulted in a decrease of D. bruxellensis cell viability almost three times higher than of S. cerevisiae, which was only slightly affected by all cell treatments. The initial viability of the treated cells was restored within 8 h of growth in sugar cane juice, with the exception of the combined treatment for D. bruxellensis. The ethanol-based cell treatment, in despite of slowing the fermentation, could decrease and maintain D. bruxellensis population under control while S. cerevisiae was taking over the fermentation along six fermentative cycles. These results indicate that it may be possible to control the growth of D. bruxellensis without major effects on S. cerevisiae. The cells could be treated between the fermentation cycles by the parcelled addition of 13 % ethanol to the tanks in which the yeast cream is treated with sulphuric acid at pH 2.0.     

Kinetics of growth and sugar consumption in yeasts

An overview is presented of the steady- and transient state kinetics of growth and formation of metabolic byproducts in yeasts.Saccharomyces cerevisiae is strongly inclined to perform alcoholic fermentation. Even under fully aerobic conditions, ethanol is produced by this yeast when sugars are present in excess. This so-called Crabtree effect probably results from a multiplicity of factors, including the mode of sugar transport and the regulation of enzyme activities involved in respiration and alcoholic fermentation. The Crabtree effect inS. cerevisiae is not caused by an intrinsic inability to adjust its respiratory activity to high glycolytic fluxes. Under certain cultivation conditions, for example during growth in the presence of weak organic acids, very high respiration rates can be achieved by this yeast.S. cerevisiae is an exceptional yeast since, in contrast to most other species that are able to perform alcoholic fermentation, it can grow under strictly anaerobic conditions.Non-Saccharomyces yeasts require a growth-limiting supply of oxygen (i.e. oxygen-limited growth conditions) to trigger alcoholic fermentation. However, complete absence of oxygen results in cessation of growth and therefore, ultimately, of alcoholic fermentation. Since it is very difficult to reproducibly achieve the right oxygen dosage in large-scale fermentations, non-Saccharomyces yeasts are therefore not suitable for large-scale alcoholic fermentation of sugar-containing waste streams. In these yeasts, alcoholic fermentation is also dependent on the type of sugar. For example, the facultatively fermentative yeastCandida utilis does not ferment maltose, not even under oxygen-limited growth conditions, although this disaccharide supports rapid oxidative growth.     

The fermentation of sugarcane molasses by Dekkera bruxellensis and the mobilization of reserve carbohydrates

The yeast Dekkera bruxellensis is considered to be very well adapted to industrial environments, in Brazil, USA, Canada and European Countries, when different substrates are used in alcoholic fermentations. Our previous study described its fermentative profile with a sugarcane juice substrate. In this study, we have extended its physiological evaluation to fermentation situations by using sugarcane molasses as a substrate to replicate industrial working conditions. The results have confirmed the previous reports of the low capacity of D. bruxellensis cells to assimilate sucrose, which seems to be the main factor that can cause a bottleneck in its use as fermentative yeast. Furthermore, the cells of D. bruxellensis showed a tendency to deviate most of sugar available for biomass and organic acids (lactic and acetic) compared with Saccharomyces cerevisiae, when calculated on the basis of their respective yields. As well as this, the acetate production from molasses medium by both yeasts was in marked contrast with the previous data on sugarcane juice. Glycerol and ethanol production by D. bruxellensis cells achieved levels of 33 and 53 % of the S. cerevisiae, respectively. However, the ethanol yield was similar for both yeasts. It is worth noting that this yeast did not accumulate trehalose when the intracellular glycogen content was 30 % lower than in S. cerevisiae. The lack of trehalose did not affect yeast viability under fermentation conditions. Thus, the adaptive success of D. bruxellensis under industrial fermentation conditions seems to be unrelated to the production of these reserve carbohydrates.     

Homofermentative Lactate Production Cannot Sustain Anaerobic Growth of Engineered Saccharomyces cerevisiae: Possible Consequence of Energy-Dependent Lactate Export

Due to a growing market for the biodegradable and renewable polymer polylactic acid, the world demand for lactic acid is rapidly increasing. The tolerance of yeasts to low pH can benefit the process economy of lactic acid production by minimizing the need for neutralizing agents. Saccharomyces cerevisiae (CEN.PK background) was engineered to a homofermentative lactate-producing yeast via deletion of the three genes encoding pyruvate decarboxylase and the introduction of a heterologous lactate dehydrogenase (EC 1.1.1.27). Like all pyruvate decarboxylase-negative S. cerevisiae strains, the engineered strain required small amounts of acetate for the synthesis of cytosolic acetyl-coenzyme A. Exposure of aerobic glucose-limited chemostat cultures to excess glucose resulted in the immediate appearance of lactate as the major fermentation product. Ethanol formation was absent. However, the engineered strain could not grow anaerobically, and lactate production was strongly stimulated by oxygen. In addition, under all conditions examined, lactate production by the engineered strain was slower than alcoholic fermentation by the wild type. Despite the equivalence of alcoholic fermentation and lactate fermentation with respect to redox balance and ATP generation, studies on oxygen-limited chemostat cultures showed that lactate production does not contribute to the ATP economy of the engineered yeast. This absence of net ATP production is probably due to a metabolic energy requirement (directly or indirectly in the form of ATP) for lactate export.     

Starvation Response of Saccharomyces cerevisiae Grown in Anaerobic Nitrogen- or Carbon-Limited Chemostat Cultures

Anaerobic starvation conditions are frequent in industrial fermentation and can affect the performance of the cells. In this study, the anaerobic carbon or nitrogen starvation response of Saccharomyces cerevisiae was investigated for cells grown in anaerobic carbon or nitrogen-limited chemostat cultures at a dilution rate of 0.1 h⁻¹ at pH 3.25 or 5. Lactic or benzoic acid was present in the growth medium at different concentrations, resulting in 16 different growth conditions. At steady state, cells were harvested and then starved for either carbon or nitrogen for 24 h under anaerobic conditions. We measured fermentative capacity, glucose uptake capacity, intracellular ATP content, and reserve carbohydrates and found that the carbon, but not the nitrogen, starvation response was dependent upon the previous growth conditions. All cells subjected to nitrogen starvation retained a large portion of their initial fermentative capacity, independently of previous growth conditions. However, nitrogen-limited cells that were starved for carbon lost almost all their fermentative capacity, while carbon-limited cells managed to preserve a larger portion of their fermentative capacity during carbon starvation. There was a positive correlation between the amount of glycogen before carbon starvation and the fermentative capacity and ATP content of the cells after carbon starvation. Fermentative capacity and glucose uptake capacity were not correlated under any of the conditions tested. Thus, the successful adaptation to sudden carbon starvation requires energy and, under anaerobic conditions, fermentable endogenous resources. In an industrial setting, carbon starvation in anaerobic fermentations should be avoided to maintain a productive yeast population.     

The Fate of Acetic Acid during Glucose Co-Metabolism by the Spoilage Yeast Zygosaccharomyces bailii

Zygosaccharomyces bailii is one of the most widely represented spoilage yeast species, being able to metabolise acetic acid in the presence of glucose. To clarify whether simultaneous utilisation of the two substrates affects growth efficiency, we examined growth in single- and mixed-substrate cultures with glucose and acetic acid. Our findings indicate that the biomass yield in the first phase of growth is the result of the weighted sum of the respective biomass yields on single-substrate medium, supporting the conclusion that biomass yield on each substrate is not affected by the presence of the other at pH 3.0 and 5.0, at least for the substrate concentrations examined. In vivo ¹³C-NMR spectroscopy studies showed that the gluconeogenic pathway is not operational and that [2−¹³C]acetate is metabolised via the Krebs cycle leading to the production of glutamate labelled on C₂, C₃ and C₄. The incorporation of [U-¹⁴C]acetate in the cellular constituents resulted mainly in the labelling of the protein and lipid pools 51.5% and 31.5%, respectively. Overall, our data establish that glucose is metabolised primarily through the glycolytic pathway, and acetic acid is used as an additional source of acetyl-CoA both for lipid synthesis and the Krebs cycle. This study provides useful clues for the design of new strategies aimed at overcoming yeast spoilage in acidic, sugar-containing food environments. Moreover, the elucidation of the molecular basis underlying the resistance phenotype of Z. bailii to acetic acid will have a potential impact on the improvement of the performance of S. cerevisiae industrial strains often exposed to acetic acid stress conditions, such as in wine and bioethanol production.     

Oxygen Consumption by Anaerobic Saccharomyces cerevisiae under Enological Conditions: Effect on Fermentation Kinetics

The anaerobic growth of the yeast Saccharomyces cerevisiae normally requires the addition of molecular oxygen, which is used to synthesize sterols and unsaturated fatty acids (UFAs). A single oxygen pulse can stimulate enological fermentation, but the biochemical pathways involved in this phenomenon remain to be elucidated. We showed that the addition of oxygen (0.3 to 1.5 mg/g [dry mass] of yeast) to a lipid-depleted medium mainly resulted in the synthesis of the sterols and UFAs required for cell growth. However, the addition of oxygen during the stationary phase in a medium containing excess ergosterol and oleic acid increased the specific fermentation rate, increased cell viability, and shortened the fermentation period. Neither the respiratory chain nor de novo protein synthesis was required for these medium- and long-term effects. As de novo lipid synthesis may be involved in ethanol tolerance, we studied the effect of oxygen addition on sterol and UFA auxotrophs (erg1 and ole1 mutants, respectively). Both mutants exhibited normal anaerobic fermentation kinetics. However, only the ole1 mutant strain responded to the oxygen pulse during the stationary phase, suggesting that de novo sterol synthesis is required for the oxygen-induced increase of the specific fermentation rate. In conclusion, the sterol pathway appears to contribute significantly to the oxygen consumption capacities of cells under anaerobic conditions. Nevertheless, we demonstrated the existence of alternative oxygen consumption pathways that are neither linked to the respiratory chain nor linked to heme, sterol, or UFA synthesis. These pathways dissipate the oxygen added during the stationary phase, without affecting the fermentation kinetics.     

Oxygen requirements of yeasts

Type species of 75 yeast genera were examined for their ability to grow anaerobically in complex and mineral media. To define anaerobic conditions, we added a redox indicator, resazurin, to the media to determine low redox potentials. All strains tested were capable of fermenting glucose to ethanol in oxygen-limited shake-flask cultures, even those of species generally regarded as nonfermentative. However, only 23% of the yeast species tested grew under anaerobic conditions. A comparative study with a number of selected strains revealed that Saccharomyces cerevisiae stands out as a yeast capable of rapid growth at low redox potentials. Other yeasts, such as Torulaspora delbrueckii and Candida tropicalis, grew poorly mu max, 0.03 and 0.05 h-1, respectively) under anaerobic conditions in mineral medium supplemented with Tween 80 and ergosterol. The latter organisms grew rapidly under oxygen limitation and then displayed a high rate of alcoholic fermentation. It can be concluded that these yeasts have hitherto-unidentified oxygen requirements for growth.     

Overexpression of <Emphasis Type="Italic">ADH1</Emphasis> and <Emphasis Type="Italic">HXT1</Emphasis> genes in the yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> improves the fermentative efficiency during tequila elaboration

This work assessed the effect of the overexpression of ADH1 and HXT1 genes in the Saccharomyces cerevisiae AR5 strain during fermentation of Agave tequilana Weber blue variety must. Both genes were cloned individually and simultaneously into a yeast centromere plasmid. Two transformant strains overexpressing ADH1 and HXT1 individually and one strain overexpressing both genes were randomly selected and named A1, A3 and A5 respectively. Overexpression effect on growth and ethanol production of the A1, A3 and A5 strains was evaluated in fermentative conditions in A. tequilana Weber blue variety must and YPD medium. During growth in YPD and Agave media, all the recombinant strains showed lower cell mass formation than the wild type AR5 strain. Adh enzymatic activity in the recombinant strains A1 and A5 cultivated in A. tequilana and YPD medium was higher than in the wild type. The overexpression of both genes individually and simultaneously had no significant effect on ethanol formation; however, the fermentative efficiency of the A5 strain increased from 80.33% to 84.57% and 89.40% to 94.29% in YPD and Agave medium respectively.     

Oxygen requirements of yeasts.

Type species of 75 yeast genera were examined for their ability to grow anaerobically in complex and mineral media. To define anaerobic conditions, we added a redox indicator, resazurin, to the media to determine low redox potentials. All strains tested were capable of fermenting glucose to ethanol in oxygen-limited shake-flask cultures, even those of species generally regarded as nonfermentative. However, only 23% of the yeast species tested grew under anaerobic conditions. A comparative study with a number of selected strains revealed that Saccharomyces cerevisiae stands out as a yeast capable of rapid growth at low redox potentials. Other yeasts, such as Torulaspora delbrueckii and Candida tropicalis, grew poorly mu max, 0.03 and 0.05 h-1, respectively) under anaerobic conditions in mineral medium supplemented with Tween 80 and ergosterol. The latter organisms grew rapidly under oxygen limitation and then displayed a high rate of alcoholic fermentation. It can be concluded that these yeasts have hitherto-unidentified oxygen requirements for growth.     

Evolution of Yeasts and Lactic Acid Bacteria During Fermentation and Storage of Bordeaux Wines

The levels of yeasts and lactic acid bacteria that naturally developed during the vinification of two red and two white Bordeaux wines were quantitatively examined. Yeasts of the genera Rhodotorula, Pichia, Candida, and Metschnikowia occurred at low levels in freshly extracted grape musts but died off as soon as fermentation commenced. Kloeckera apiculata (Hanseniaspora uvarum), Torulopsis stellata, and Saccharomyces cerevisiae, the dominant yeasts in musts, proliferated to conduct alcoholic fermentation. K. apiculata and eventually T. stellata died off as fermentation progressed, leaving S. cerevisiae as the dominant yeast until the termination of fermentation by the addition of sulfur dioxide. At least two different strains of S. cerevisiae were involved in the fermentation of one of the red wines. Low levels of lactic acid bacteria (Pediococcus cerevisiae, Leuconostoc mesenteroides, and Lactobacillus spp.) were present in grape musts but died off during alcoholic fermentation. The malolactic fermentation developed in both red wines soon after alcoholic fermentation and correlated with the vigorous growth of at least three different strains of Leuconostoc oenos.     

Effect of Specific Growth Rate on Fermentative Capacity of Baker’s Yeast

The specific growth rate is a key control parameter in the industrial production of baker’s yeast. Nevertheless, quantitative data describing its effect on fermentative capacity are not available from the literature. In this study, the effect of the specific growth rate on the physiology and fermentative capacity of an industrial Saccharomyces cerevisiae strain in aerobic, glucose-limited chemostat cultures was investigated. At specific growth rates (dilution rates, D) below 0.28 h⁻¹, glucose metabolism was fully respiratory. Above this dilution rate, respirofermentative metabolism set in, with ethanol production rates of up to 14 mmol of ethanol · g of biomass⁻¹ · h⁻¹ at D = 0.40 h⁻¹. A substantial fermentative capacity (assayed offline as ethanol production rate under anaerobic conditions) was found in cultures in which no ethanol was detectable (D < 0.28 h⁻¹). This fermentative capacity increased with increasing dilution rates, from 10.0 mmol of ethanol · g of dry yeast biomass⁻¹ · h⁻¹ at D = 0.025 h⁻¹ to 20.5 mmol of ethanol · g of dry yeast biomass⁻¹ · h⁻¹ at D = 0.28 h⁻¹. At even higher dilution rates, the fermentative capacity showed only a small further increase, up to 22.0 mmol of ethanol · g of dry yeast biomass⁻¹ · h⁻¹ at D = 0.40 h⁻¹. The activities of all glycolytic enzymes, pyruvate decarboxylase, and alcohol dehydrogenase were determined in cell extracts. Only the in vitro activities of pyruvate decarboxylase and phosphofructokinase showed a clear positive correlation with fermentative capacity. These enzymes are interesting targets for overexpression in attempts to improve the fermentative capacity of aerobic cultures grown at low specific growth rates.The quality of commercial baker’s yeast (Saccharomyces cerevisiae) is determined by many parameters, including storage stability, osmotolerance, freeze-thaw resistance, rehydration resistance of dried yeast, and color. In view of the primary role of baker’s yeast in dough, fermentative capacity (i.e., the specific rate of carbon dioxide production by yeast upon its introduction into dough) is a particularly important parameter (2).In S. cerevisiae, high sugar concentrations and high specific growth rates trigger alcoholic fermentation, even under fully aerobic conditions (6, 18). Alcoholic fermentation during the industrial production of baker’s yeast is highly undesirable, as it reduces the biomass yield on the carbohydrate feedstock. Industrial baker’s yeast production is therefore performed in aerobic, sugar-limited fed-batch cultures. The conditions in such cultures differ drastically from those in the dough environment, which is anaerobic and with sugars at least initially present in excess (23).Optimization of biomass productivity requires that the specific growth rate and biomass yield in the fed-batch process be as high as possible. In the early stage of the process, the maximum feasible growth rate is dictated by the threshold specific growth rate at which respirofermentative metabolism sets in. In later stages, the specific growth rate is decreased to avoid problems with the limited oxygen transfer and/or cooling capacity of industrial bioreactors (10, 27). The actual growth rate profile during fed-batch cultivation is controlled primarily by the feed rate profile of the carbohydrate feedstock (4, 22). Generally, an initial exponential feed phase is followed by phases with constant and declining feed rates, respectively (8).From a theoretical point of view, the objective of suppressing alcoholic fermentation during the production phase may interfere with the aim of obtaining a high fermentative capacity in the final product. Process optimization has so far been based on strain selection and on empirical optimization of environmental conditions during fed-batch cultivation (e.g., pH, temperature, aeration rate, and feed profiles of sugar, nitrogen, and phosphorus [5, 10, 23]). For rational optimization of the specific growth rate profile, knowledge of the relation between specific growth rate and fermentative capacity is of primary importance. Nevertheless, quantitative data on this subject cannot be found in the literature.The chemostat cultivation system allows manipulation of the specific growth rate (which is equal to the dilution rate) while keeping other important growth conditions constant. Similar to industrial fed-batch cultivation, sugar-limited chemostat cultivation allows fully respiratory growth of S. cerevisiae on sugars (21, 37, 39). This is not possible in batch cultures, which by definition require high sugar concentrations, which lead to alcoholic fermentation, even during aerobic growth (6, 18, 37). Thus, as an experimental system, batch cultures bear little resemblance to the aerobic baker’s yeast production process. Indeed, we have recently shown that differences in fermentative capacity between a laboratory strain of S. cerevisiae and an industrial strain became apparent only in glucose-limited chemostat cultures but not in batch cultures (30).The aim of the present study was to assess the effect of specific growth rate on fermentative capacity in an industrial baker’s yeast strain grown in aerobic, sugar-limited chemostat cultures. Furthermore, the effect of specific growth rate on in vitro activities of key glycolytic and fermentative enzymes was investigated in an attempt to identify correlations between fermentative capacity and enzyme levels.     

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.     

Changes in the intracellular concentrations of the adenosine phosphates and nicotinamide adenine dinucleotides ofSaccharomyces cerevisiae during batch fermentation

Significant changes in the intracellular concentrations of adenosine phosphates and nicotinamide adenine dinucleotides were observed during fermentation of grape must by three different strains ofSaccharomyces cerevisiae: S. cerevisiae var.cerevisiae, a typical fermentative yeast strain and two flor-veil-forming strains,S. cerevisiae var.bayanus andS. cerevisiae var.capensis. The intracellular concentration of ATP was always higher inS. cerevisiae var.cerevisiae than in the flor-veil-forming strains. NAD⁺ and NADP⁺ concentrations decreased at faster rates in the flor-veil-forming yeasts than in the other yeast but NADH concentration was the same in all yeasts for the first 10 days of fermentation. NADPH concentration was always lower inS. cerevisiae var.cerevisiae than in the other yeasts and this yeast also showed higher rates of growth and fermentation during the early stages of the fermentation and the presence of non-viable cells at the end of fermentation. In contrast, the flor-veil-forming strains maintained growth and fermentation capabilities for a relatively long time and viable cells were present throughout the entire fermentation process (31 days).The authors are with the Department of Microbiology, Faculty of Sciences, University of Cordoba, Avda. San Alberto Magno s/n, 14004-Córdoba, Spain     

Benzoic Acid, a Weak Organic Acid Food Preservative, Exerts Specific Effects on Intracellular Membrane Trafficking Pathways in Saccharomyces cerevisiae

Microbial spoilage of food causes losses of up to 40% of all food grown for human consumption worldwide. Yeast growth is a major factor in the spoilage of foods and beverages that are characterized by a high sugar content, low pH, and low water activity, and it is a significant economic problem. While growth of spoilage yeasts such as Zygosaccharomyces bailii and Saccharomyces cerevisiae can usually be retarded by weak organic acid preservatives, the inhibition often requires levels of preservative that are near or greater than the legal limits. We identified a novel synergistic effect of the chemical preservative benzoic acid and nitrogen starvation: while exposure of S. cerevisiae to either benzoic acid or nitrogen starvation is cytostatic under our conditions, the combination of the two treatments is cytocidal and can therefore be used beneficially in food preservation. In yeast, as in all eukaryotic organisms, survival under nitrogen starvation conditions requires a cellular response called macroautophagy. During macroautophagy, cytosolic material is sequestered by intracellular membranes. This material is then targeted for lysosomal degradation and recycled into molecular building blocks, such as amino acids and nucleotides. Macroautophagy is thought to allow cellular physiology to continue in the absence of external resources. Our analyses of the effects of benzoic acid on intracellular membrane trafficking revealed that there was specific inhibition of macroautophagy. The data suggest that the synergism between nitrogen starvation and benzoic acid is the result of inhibition of macroautophagy by benzoic acid and that a mechanistic understanding of this inhibition should be beneficial in the development of novel food preservation technologies.