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.     

Zygosaccharomyces lentus: a significant new osmophilic, preservative-resistant spoilage yeast, capable of growth at low temperature

Zygosaccharomyces lentus is a yeast species recently identified from its physiology and 18S ribosomal sequencing (Steels et al. 1999).The physiological characteristics of five strains of this new yeast so far isolated were investigated, particularly those of technical significance for a spoilage yeast, namely temperature range, pH range, osmotolerance, sugar fermentation, resistance to food preservatives such as sorbic acid, benzoic acid and dimethyldicarbonate (DMDC; Velcorin). Adaptation to benzoic acid, and growth in shaking and static culture were also investigated. Zygosaccharomyces lentus strains grew over a wide range of temperature (4-25 degrees C) and pH 2.2-7.0. Growth at 4 degrees C was significant. Zygosaccharomyces lentus strains grew at 25-26 degrees C in static culture but were unable to grow in aerobic culture close to their temperature maximum. All Z. lentus strains grew in 60% w/v sugar and consequently, are osmotolerant. Zygosaccharomyces lentus strains could utilize sucrose, glucose or fructose as a source of fermentable sugar, but not galactose. Zygosaccharomyces lentus strains were resistant to food preservatives, growing in sorbic acid up to 400 mg l-1 and benzoic acid to 900 mg l-1 at pH 4.0. Adaptation to higher preservative concentrations was demonstrated with benzoic acid. Resistance to DMDC was shown to be greater than that of Z. bailii and Saccharomyces cerevisiae. This study confirms that Z. lentus is an important food spoilage organism potentially capable of growth in a wide range of food products, particularly low pH, high sugar foods and drinks. It is likely to be more significant than Z. bailii in the spoilage of chilled products.     

Heterologous protein production in Zygosaccharomyces bailii: physiological effects and fermentative strategies

The optimisation and scale-up of a specific protein production process have to take into account cultivation conditions as well as cell physiology of growth and the influence of foreign protein expression on host cell metabolism. The ability of Zygosaccharomyces bailii to tolerate high sugar concentrations as well as high temperatures and acidic environments renders this "non-conventional" yeast suitable for the development of biotechnological processes like heterologous protein production. This work addresses the production of human interleukin-1beta by a recombinant Z. bailii strain. We found that the heterologous protein production causes some modifications of the Z. bailii carbon metabolism, leading to a reduced biomass yield. The other important factor is the dependence of the recombinant IL-1beta production/secretion on the growth rate. Among the cultivation strategies studied, the most appropriate in terms of production and productivity was the fed-batch mode.     

Glucose respiration and fermentation in Zygosaccharomyces bailii and Saccharomyces cerevisiae express different sensitivity patterns to ethanol and acetic acid

In the yeast Zygosaccharomyces bailii ISA 1307, respiration and fermentation ofglucose were exponentially inhibited by ethanol, both processes displaying similar sensitivity tothe alcohol. Moreover, the degree of inhibition on fermentation was of the same magnitude as thatreported for Saccharomyces cerevisiae. Acetic acid also inhibited these two metabolicprocesses in Z. bailii , with the kinetics of inhibition again being exponential. However,inhibition of fermentation was much less pronounced than in S. cerevisiae. The valuesestimated with Z. bailii for the minimum inhibitory concentration of acetic acid rangedfrom 100 to 240 mmol l⁻¹ total acetic acid compared with values of near zeroreported for S. cerevisiae. The inhibitory effects of acetic acid on Z. bailii were notsignificantly potentiated by ethanol.     

Steady-state and transient-state analyses of aerobic fermentation in Saccharomyces kluyveri

Some yeasts, such as Saccharomyces cerevisiae, produce ethanol at fully aerobic conditions, whereas other yeasts, such as Kluyveromyces lactis, do not. In this study we investigated the occurrence of aerobic alcoholic fermentation in the petite-negative yeast Saccharomyces kluyveri that is only distantly related to S. cerevisiae. In aerobic glucose-limited continuous cultures of S. kluyveri, two growth regimens were observed: at dilution rates below 0.5 h(-1) the metabolism was purely respiratory, and at dilution rates above 0.5 h(-1) the metabolism was respiro-fermentative. The dilution rate at which the switch in metabolism occurred, i.e. the critical dilution rate, was 66% higher than the typical critical dilution rate of S. cerevisiae. The maximum specific oxygen consumption rate around the critical dilution rate was found to 13.6 mmol (g dry weight)(-1) h(-1) and the capacity of the pyruvate dehydrogenase-bypass pathway was estimated to be high from in vitro enzyme activities; especially the specific activity of acetyl-CoA synthetase was much higher than in S. cerevisiae at all tested conditions. Addition of glucose to respiring cells of S. kluyveri led to ethanol formation after a delay of 20-50 min (depending on culture conditions prior to the pulse), which is in contrast to S. cerevisiae that ferments immediately after glucose addition.     

Transport of acetic acid in Zygosaccharomyces bailii: effects of ethanol and their implications on the resistance of the yeast to acidic environments.

Cells of Zygosaccharomyces bailii ISA 1307 grown in a medium with acetic acid, ethanol, or glycerol as the sole carbon and energy source transported acetic acid by a saturable transport system. This system accepted propionic and formic acids but not lactic, sorbic, and benzoic acids. When the carbon source was glucose or fructose, the cells displayed activity of a mediated transport system specific for acetic acid, apparently not being able to recognize other monocarboxylic acids. In both types of cells, ethanol inhibited the transport of labelled acetic acid. The inhibition was noncompetitive, and the dependence of the maximum transport rate on the ethanol concentration was found to be exponential. These results reinforced the belief that, under the referenced growth conditions, the acid entered the cells mainly through a transporter protein. The simple diffusion of the undissociated acid appeared to contribute, with a relatively low weight, to the overall acid uptake. It was concluded that in Z. bailii, ethanol plays a protective role against the possible negative effects of acetic acid by inhibiting its transport and accumulation. Thus, the intracellular concentration of the acid could be maintained at levels lower than those expected if the acid entered the cells only by simple diffusion.     

Adaptive response to acetic acid in the highly resistant yeast species Zygosaccharomyces bailii revealed by quantitative proteomics

Zygosaccharomyces bailii is the most tolerant yeast species to acetic acid-induced toxicity, being able to grow in the presence of concentrations of this food preservative close to the legal limits. For this reason, Z. bailii is the most important microbial contaminant of acidic food products but the mechanisms behind this intrinsic resistance to acetic acid are very poorly characterized. To gain insights into the adaptive response and tolerance to acetic acid in Z. bailii, we explored an expression proteomics approach, based on quantitative 2DE, to identify alterations occurring in the protein content in response to sudden exposure or balanced growth in the presence of an inhibitory but nonlethal concentration of this weak acid. A coordinate increase in the content of proteins involved in cellular metabolism, in particular, in carbohydrate metabolism (Mdh1p, Aco1p, Cit1p, Idh2p, and Lpd1p) and energy generation (Atp1p and Atp2p), as well as in general and oxidative stress response (Sod2p, Dak2p, Omp2p) was registered. Results reinforce the concept that glucose and acetic acid are coconsumed in Z. bailii, with acetate being channeled into the tricarboxylic acid cycle. When acetic acid is the sole carbon source, results suggest the activation of gluconeogenic and pentose phosphate pathways, based on the increased content of several proteins of these pathways after glucose exhaustion.     

Reduction of volatile acidity of wines by selected yeast strains

Herein, we isolate and characterize wine yeasts with the ability to reduce volatile acidity of wines using a refermentation process, which consists in mixing the acidic wine with freshly crushed grapes or musts or, alternatively, in the incubation with the residual marc. From a set of 135 yeast isolates, four strains revealed the ability to use glucose and acetic acid simultaneously. Three of them were identified as Saccharomyces cerevisiae and one as Lachancea thermotolerans. Among nine commercial S. cerevisiae strains, strains S26, S29, and S30 display similar glucose and acetic acid initial simultaneous consumption pattern and were assessed in refermentation assays. In a medium containing an acidic wine with high glucose-low ethanol concentrations, under low oxygen availability, strain S29 is the most efficient one, whereas L. thermotolerans 44C is able to decrease significantly acetic acid similar to the control strain Zygosaccharomyces bailii ISA 1307 but only under aerobic conditions. Conversely, for low glucose-high ethanol concentrations, under aerobic conditions, S26 is the most efficient acid-degrading strain, while under limited-aerobic conditions, all the S. cerevisiae strains studied display acetic acid degradation efficiencies identical to Z. bailii. Moreover, S26 strain also reveals capacity to decrease volatile acidity of wines. Together, the S. cerevisiae strains characterized herein appear promising for the oenological removal of volatile acidity of acidic wines.     

Factors influencing the utilisation of l-malate by yeasts

The utilisation of L-malate and the effect of glucose concentration on malate utilisation under semi-anaerobic conditions were investigated in three yeasts unable to grow on malate as sole carbon source (Saccharomyces cerevisiae, Schizosaccharomyces malidevorans, Zygosaccharomyces bailii) and two yeasts able to utilise the TCA cycle intermediate as sole carbon source (Pichia stipitis and Pachysolen tannophilus). Utilisation of malate by both Schiz. malidevorans and Z. bailii was reduced at high and low levels of glucose. In the absence of glucose, P. stipitis and Pa. tannophilus utilised malate rapidly; however, their utilisation was drastically reduced in the presence of glucose, suggesting that malate utilisation is under catabolite repression.     

Cybernetic model of the growth dynamics of Saccharomyces cerevisiae in batch and continuous cultures.

Growth of Saccharomyces cerevisiae on glucose in aerobic batch culture follows the well-documented diauxic pattern of completely fermenting glucose to ethanol during the first exponential growth phase, followed by an intermediate lag phase and a second exponential growth phase consuming ethanol. In continuous cultures over a range of intermediate dilution rates, the yeast bioreactor exhibits sustained oscillations in all the measured concentrations, such as cell mass, glucose, ethanol, and dissolved oxygen, the amounts of intracellular storage carbohydrates, such as glycogen and trehalose, the fraction of budded cells as well as the culture pH. We present here a structured, unsegregated model for the yeast growth dynamics developed from the 'cybernetic' modeling framework, to simulate the dynamic competition between all the available metabolic pathways. This cybernetic model accurately predicts all the key experimentally observed aspects: (i) in batch cultures, duration of the intermediate lag phase, sequential production and consumption of ethanol, and the dynamics of the gaseous exchange rates of oxygen and carbon dioxide; and (ii) in continuous cultures, the spontaneous generation of oscillations as well as the variations in period and amplitude of oscillations when the dilution rate or agitatin rate are changed.     

Advances in molecular tools for the use of Zygosaccharomyces bailii as host for biotechnological productions and construction of the first auxotrophic mutant

The nonconventional yeast Zygosaccharomyces bailii has been proposed as a new host for biotechnological processes due to convenient properties such as its resistance to high sugar concentrations, relatively high temperatures and especially to acidic environments. We describe a series of new expression vectors specific for Z. bailii and the resulting improvements in production levels. By exploiting the sequences of the endogenous plasmid pSB2, 2microm-like multicopy vectors were obtained, giving a fivefold increase in production. A specific integrative vector was developed which led to 100% stability in the absence of selective pressure; a multiple-integration vector was constructed, based on an rRNA gene unit portion cloned and sequenced for this purpose, driving the insertion of up to 80 copies of the foreign construct. Moreover, we show the construction of the first stable auxotrophic mutant of Z. bailii, obtained by targeted gene deletion applied to ZbLEU2. The development of molecular tools for the Z. bailii manipulation has now reached a level that may be compatible with its industrial exploitation; the production of organic acids is a prominent field of application.     

Steady-state and transient-state analysis of growth and metabolite production in a Saccharomyces cerevisiae strain with reduced pyruvate-decarboxylase activity

Pyruvate decarboxylase is a key enzyme in the production of low-molecular-weight byproducts (ethanol, acetate) in biomass-directed applications of Saccharomyces cerevisiae. To investigate whether decreased expression levels of pyruvate decarboxylase can reduce byproduct formation, the PDC2 gene, which encodes a positive regulator of pyruvate-decarboxylase synthesis, was inactivated in the prototrophic strain S. cerevisiae CEN. PK113-7D. This caused a 3-4-fold reduction of pyruvate-decarboxylase activity in glucose-limited, aerobic chemostat cultures grown at a dilution rate of 0.10 h(-1). Upon exposure of such cultures to a 50 mM glucose pulse, ethanol and acetate were the major byproducts formed by the wild type. In the pdc2Delta strain, formation of ethanol and acetate was reduced by 60-70%. In contrast to the wild type, the pdc2Delta strain produced substantial amounts of pyruvate after a glucose pulse. Nevertheless, its overall byproduct formation was ca. 50% lower. The specific rate of glucose consumption after a glucose pulse to pdc2Delta cultures was about 40% lower than in wild-type cultures. This suggests that, at reduced pyruvate-decarboxylase activities, glycolytic flux is controlled by NADH reoxidation. In aerobic, glucose-limited chemostat cultures, the wild type exhibited a mixed respiro-fermentative metabolism at dilution rates above 0.30 h(-1). Below this dilution rate, sugar metabolism was respiratory. At dilution rates up to 0.20 h(-1), growth of the pdc2Delta strain was respiratory and biomass yields were similar to those of wild-type cultures. Above this dilution rate, washout occurred. The low micro(max) of the pdc2Delta strain in glucose-limited chemostat cultures indicates that occurrence of respiro-fermentative metabolism in wild-type cultures is not solely caused by competition of respiration and fermentation for pyruvate. Furthermore, it implies that inactivation of PDC2 is not a viable option for reducing byproduct formation in industrial fermentations.     

The production and growth characteristics of yeast and mycelial forms of Candida albicans in continuous culture.

The growth characteristics of Candida albicans CM145,348 have been examined under aerobic conditions in continuous culture. At different steady states the environment was controlled with respect to the concentrations of dissolved oxygen, carbon and nitrogen, the pH, and the temperature. Dry matter, substrate concentration, yield, specific oxygen uptake, specific carbon dioxide release and respiration quotient were examined as a function of the dilution rate. The morphology depended on the carbon source. Maltose produced a mycelial morphology, whereas with lactate a yeast culture was obtained. With fructose or glucose as a carbon source a mixed morphology of yeast, pseudo-mycelial and mycelial forms was produced. A larger number of different growth conditions were examined in batch culture but a mixed morphology was always obtained.     

Modeling of Xanthophyllomyces dendrorhous growth on glucose and overflow metabolism in batch and fed-batch cultures for astaxanthin production

An astaxanthin-producing yeast Xanthophyllomyces dendrorhous ENM5 was cultivated in a liquid medium containing 50 g/L glucose as the major carbon source in stirred fermentors (1.5-L working volume) in fully aerobic conditions. Ethanol was produced during the exponential growth phase as a result of overflow metabolism or fermentative catabolism of glucose by yeast cells. After accumulating to a peak of 3.5 g/L, the ethanol was consumed by yeast cells as a carbon source when glucose in the culture was nearly exhausted. High initial glucose concentrations and ethanol accumulation in the culture had inhibitory effects on cell growth. Astaxanthin production was partially associated with cell growth. Based on these culture characteristics, we constructed a modified Monod kinetic model incorporating substrate (glucose) and product (ethanol) inhibition to describe the relationship of cell growth rate with glucose and ethanol concentrations. This kinetic model, coupled with the Luedeking-Piret equation for the astaxanthin production, gave satisfactory prediction of the biomass production, glucose consumption, ethanol formation and consumption, and astaxanthin production in batch cultures over 25-75 g/L glucose concentration ranges. The model was also applied to fed-batch cultures to predict the optimum feeding scheme (feeding glucose and corn steep liquor) for astaxanthin production, leading to a high volumetric yield (28.6 mg/L) and a high productivity (5.36 mg/L/day).     

酱香型白酒酿造拜耳接合酵母生理代谢特征及其与地衣芽孢杆菌相互作用

【目的】拜耳接合酵母(Zygosaccharomyces bailii)是酱香型白酒酿造过程优势菌株。通过研究拜耳接合酵母酿造相关生理代谢特征及其与酿造环境中其它功能菌株的相互作用,探索其在白酒酿造过程中的贡献。【方法】从酱香酒酿造中筛选一株性能优良的拜耳接合酵母,比较其与模式菌株(Z.bailii,ATCC 58445)的生理代谢特征。通过组合发酵研究其与产酱香特征风味细菌地衣芽孢杆菌的相互作用。【结果】从酱香型酒醅中筛选得到一株性状优良的拜耳接合酵母(Z.bailii 15),可耐受p H 2.0和37°C高温及8%酒精浓度(体积比),较模式株更适应酿造环境,酒精产量(33.58 g/L)也远高于模式株(19.04 g/L),且与酱香型白酒酿酒酵母MT1(34.29 g/L)相当。该菌可产多种风味物质,与模式株相比,独特产生法呢醇、十二醇、2-壬醇、2-乙基己醇、癸酸、月桂酸、辛酸、辛酸乙酯、苯乙酮、4-叔丁基苯酚。共培养体系中,30°C条件下地衣芽孢杆菌对拜耳接合酵母生长影响不大,而在37°C地衣芽孢杆菌抑制拜耳接合酵母生长。此外,地衣芽孢杆菌对拜耳接合酵母乙醇转化率有促进作用,共培养体系风味物质种类及含量也都受到很大影响。【结论】拜耳接合酵母在酱香型白酒酿造体系中产酒精、产风味方面表现优异,对酱香型白酒生产具有重要贡献。     

Kinetics of biphasic growth of yeast in continuous and fed-batch cultures

The influence of dilution rate on the production of biomass, ethanol, and invertase in an aerobic culture of Saccharomyces carlsbergensis was studied in a glucose-limited chemostat culture. A kinetic model was developed to analyze the biphasic growth of yeast on both the glucose remaining and the ethanol produced in the culture. The model assumes a double effect where glucose regulates the flux of glucose catabolism (respiration and aerobic fermentation) and the ethanol utilization in yeast cells. The model could successfully demonstrate the experimental results of a chemostat culture featuring the monotonic decrease of biomass concentration with an increase of dilution rate higher than 0.2 hr^?1 as well as the maximum ethanol concentration at a particular dilution rate around 0.5 hr^?1. Some supplementary data were collected from an ethanol-limited aerobic chemostat culture and a glucose-limited anaerobic chemostat culture to use in the model calculation. Some parametric constants of cell growth, ethanol production, and invertase formation were determined in batch cultures under aerobic and anaerobic states as summarized in a table in comparison with the chemostat data. Using the constants, a prediction of the optimal control of a glucose fed-batch yeast culture was conducted in connection with an experiment for harvesting a high yield of yeast cells with high invertase activity.     

Physiological characterisation of a pyruvate-carboxylase-negative Saccharomyces cerevisiae mutant in batch and chemostat cultures

A prototrophic pyruvate-carboxylase-negative (Pyc-) mutant was constructed by deleting the PYC1 and PYC2 genes in a CEN.PK strain of Saccharomyces cerevisiae. Its maximum specific growth rate on ethanol was identical to that of the isogenic wild type but it was unable to grow in batch cultures in glucose-ammonia media. Consistent with earlier reports, growth on glucose could be restored by supplying aspartate as a sole nitrogen source. Ethanol could not replace aspartate as a source of oxaloacetate in batch cultures. To investigate whether alleviation of glucose repression allowed expression of alternative pathways for oxaloacetate synthesis, the Pyc- strain and an isogenic wild-type strain were grown in aerobic carbon-limited chemostat cultures at a dilution rate of 0.10 h-1 on mixtures of glucose and ethanol. In such mixed-substrate chemostat cultures of the Pyc- strain, steady-state growth could only be obtained when ethanol contributed 30% or more of the substrate carbon in the feed. Attempts to further decrease the ethanol content of the feed invariably resulted in washout. In Pyc- as well as in wild-type cultures, levels of isocitrate lyase, malate synthase and phospho-enol-pyruvate carboxykinase in cell extracts decreased with a decreasing ethanol content in the feed. Nevertheless, at the lowest ethanol fraction that supported growth of the Pyc- mutant, activities of the glyoxylate cycle enzymes in cell extracts were still sufficient to meet the requirement for C4-compounds in biomass synthesis. This suggests that factors other than glucose repression of alternative routes for oxaloacetate synthesis prevent growth of Pyc-mutants on glucose.     

Effects of a hexokinase II deletion on the dynamics of glycolysis in continuous cultures of Saccharomyces cerevisiae

In glucose-limited aerobic chemostat cultures of a wild-type Saccharomyces cerevisiae and a derived hxk2 null strain, metabolic fluxes were identical. However, the concentrations of intracellular metabolites, especially fructose 1,6-bisphosphate, and hexose-phosphorylating activities differed. Interestingly, the hxk2 null strain showed a higher maximal growth rate and higher Crabtree threshold dilution rate, revealing a higher oxidative capacity for this strain. After a pulse of glucose, aerobic glucose-limited cultures of wild-type S. cerevisiae displayed an overshoot in the intracellular concentrations of glucose 6-phosphate, fructose 6-phosphate, and fructose 1,6-bisphosphate before a new steady state was established, in contrast to the hxk2 null strain which reached a new steady state without overshoot of these metabolites. At low dilution rates the overshoot of intracellular metabolites in the wild-type strain coincided with the immediate production of ethanol after the glucose pulse. In contrast, in the hxk2 null strain the production of ethanol started gradually. However, in spite of the initial differences in ethanol production and dynamic behaviour of the intracellular metabolites, the steady-state fluxes after transition from glucose limitation to glucose excess were not significantly different in the wild-type strain and the hxk2 null strain at any dilution rate.     

Reactions of Saccharomyces cerevisiae and Zygosaccharomyces bailii to sulphite

Sulphite inhibited growth of all four yeasts studied, Zygosaccharomyces bailii NCYC 563 being most sensitive and Saccharomyces cerevisiae NCYC 431 the least. Vertical Woolf-Eadie plots were obtained for initial velocities of 35S accumulation by all four yeasts suspended in high concentrations of sulphite. Equilibrium levels of 35S accumulation were reached somewhat faster with strains of S. cerevisiae than with those of Z. bailii. With all four yeasts, the greater the extent of 35S accumulation, the larger was the decline in internal pH value. Growth of S. cerevisiae TC8 and Z. bailii NCYC 563, but to a lesser extent of S. cerevisiae NCYC 431 and Z. bailii NCYC 1427, was inhibited when mid exponential-phase cultures were supplemented with 1.0 or 2.0 mM-sulphite, the decrease in growth being accompanied by a decline in ethanol production. Unless growth was completely inhibited, the sulphite-induced decline in growth was accompanied by production of acetaldehyde and additional glycerol.     

Influence of fructose and glucose on the production of exopolysaccharides and the activities of enzymes involved in the sugar metabolism and the synthesis of sugar nucleotides in Lactobacillus delbrueckii subsp. bulgaricus NCFB 2772

 The effect of fructose and glucose on the growth, production of exopolysaccharides and the activities of enzymes involved in the synthesis of sugar nucleotides in Lactobacillus delbrueckii subsp. bulgaricus grown in continuous culture was investigated. When grown on fructose, the strain produced 25 mg l^-1 exopolysaccharide composed of glucose and galactose in the ratio 1:2.4. When the carbohydrate source was switched to a mixture of fructose and glucose, the exopolysaccharide production increased to 80 mg l^-1, while the sugar composition of the exopolysaccharide changed to glucose, galactose and rhamnose in a ratio of 1:7.0:0.8. A switch to glucose as the sole carbohydrate source had no further effect. Analysis of the enzymes involved in the synthesis of sugar nucleotides indicates that in cell-free extracts of glucose-grown cells the activity of UDP-glucose pyrophosphorylase was higher than that in cell-free extracts of fructose-grown cells. The activities of dTDP-glucose pyrophosphorylase and the rhamnose synthetic enzyme system were very low in glucose-grown cultures but could not be detected in fructose-grown cultures. Cells grown on a mixture of fructose and glucose showed similar enzyme activities as cells grown on glucose. Analysis of the intracellular level of sugar nucleotides in glucose-grown cultures of L. delbrueckii subsp. bulgaricus showed the presence of UDP-glucose and UDP-galactose in a ratio of 3.3:1, respectively, a similar ratio and slightly lower concentrations were found in fructose-grown cultures. The lower production of exopolysaccharides in cultures grown on fructose may be caused by the more complex pathway involved in the synthesis of sugar nucleotides. The absence of activities of enzymes leading to the synthesis of rhamnose nucleotides in fructose-grown cultures appeared to result in the absence of rhamnose monomer in the exopolysaccharides produced on fructose. Received: 1 February 1996/Received revision: 31 May 1996/Accepted: 2 June 1996