Use of chemostat cultures mimicking different phases of wine fermentations as a tool for quantitative physiological analysis.

BACKGROUND: Saccharomyces cerevisiae is the most relevant yeast species conducting the alcoholic fermentation that takes place during winemaking. Although the physiology of this model organism has been extensively studied, systematic quantitative physiology studies of this yeast under winemaking conditions are still scarce, thus limiting the understanding of fermentative metabolism of wine yeast strains and the systematic description, modelling and prediction of fermentation processes. In this study, we implemented and validated the use of chemostat cultures as a tool to simulate different stages of a standard wine fermentation, thereby allowing to implement metabolic flux analyses describing the sequence of metabolic states of S. cerevisae along the wine fermentation. RESULTS: Chemostat cultures mimicking the different stages of standard wine fermentations of S. cerevisiae EC1118 were performed using a synthetic must and strict anaerobic conditions. The simulated stages corresponded to the onset of the exponential growth phase, late exponential growth phase and cells just entering stationary phase, at dilution rates of 0.27, 0.04, 0.007 h-1, respectively. Notably, measured substrate uptake and product formation rates at each steady state condition were generally within the range of corresponding conversion rates estimated during the different batch fermentation stages.Moreover, chemostat data were further used for metabolic flux analysis, where biomass composition data for each condition was considered in the stoichiometric model. Metabolic flux distributions were coherent with previous analyses based on batch cultivations data and the pseudo-steady state assumption. CONCLUSIONS: Steady state conditions obtained in chemostat cultures reflect the environmental conditions and physiological states of S. cerevisiae corresponding to the different growth stages of a typical batch wine fermentation, thereby showing the potential of this experimental approach to systematically study the effect of environmental relevant factors such as temperature, sugar concentration, C/N ratio or (micro) oxygenation on the fermentative metabolism of wine yeast strains.

Biomass production and alcoholic fermentation performance of Saccharomyces cerevisiae as a function of nitrogen source.

Nitrogen limitation is one of the most common causes for stuck or sluggish fermentation. A broad range of values have been reported as the minimum nitrogen concentration necessary for the completion of alcoholic fermentation. We have analyzed the minimum nitrogen concentration required to yield the maximum biomass (nitrogen reference value) using a microwell plate reader to monitor fermentation with different nitrogen sources and sugar concentrations. The biomass yield was dependent on the amount of available nitrogen, the nature of nitrogen source, and the sugar concentration in the medium. Nevertheless, achieving the maximum biomass was not sufficient to ensure the completion of the alcoholic fermentation, because the fermentation of 280 g sugar L(-1) stuck, regardless of the nature and concentration of nitrogen source. However, a mixture of five amino acids (Leu, Ile, Val, Phe and Thr) as the nitrogen source allowed for maximum sugar consumption. Analysis of cell vitality by impedance showed a significant improvement in the vitality for cells fermenting using this amino acid combination.

Oxygen availability strongly affects chronological lifespan and thermotolerance in batch cultures of Saccharomyces cerevisiae.

Stationary-phase (SP) batch cultures of Saccharomyces cerevisiae, in which growth has been arrested by carbon-source depletion, are widely applied to study chronological lifespan, quiescence and SP-associated robustness. Based on this type of experiments, typically performed under aerobic conditions, several roles of oxygen in aging have been proposed. However, SP in anaerobic yeast cultures has not been investigated in detail. Here, we use the unique capability of S. cerevisiae to grow in the complete absence of oxygen to directly compare SP in aerobic and anaerobic bioreactor cultures. This comparison revealed strong positive effects of oxygen availability on adenylate energy charge, longevity and thermotolerance during SP. A low thermotolerance of anaerobic batch cultures was already evident during the exponential growth phase and, in contrast to the situation in aerobic cultures, was not substantially increased during transition into SP. A combination of physiological and transcriptome analysis showed that the slow post-diauxic growth phase on ethanol, which precedes SP in aerobic, but not in anaerobic cultures, endowed cells with the time and resources needed for inducing longevity and thermotolerance. When combined with literature data on acquisition of longevity and thermotolerance in retentostat cultures, the present study indicates that the fast transition from glucose excess to SP in anaerobic cultures precludes acquisition of longevity and thermotolerance. Moreover, this study demonstrates the importance of a preceding, calorie-restricted conditioning phase in the acquisition of longevity and stress tolerance in SP yeast cultures, irrespective of oxygen availability.

New insights into the advantages of ammonium as a winemaking nutrient.

Nitrogen limitation is the most common cause for stuck or sluggish fermentation in winemaking, and it is usually dealt with by supplementing grape juice with either ammonium salts or organic nutrients. These practices have a direct impact on both fermentation kinetics and the sensorial features of the final product. The aim of this work is to provide a detailed characterization of yeast physiology in response to ammonium supplementation during alcoholic fermentation. This is done by determining changes in metabolic rates on a high frequency basis, as a sensitive way to detect the impact of fermentation conditions on yeast physiology. Our results indicate that the choice of supplementation strategy has an impact on several enological parameters like fermentation length, volatile acidity, final glycerol content, and aroma profile. Interestingly, a higher proportion of ammonium relates with improved glycerol and volatile acidity, for the same global yeast assimilable nitrogen content. However, ammonium over-supplementation has a negative impact on quality related parameters, notably on volatile acidity and aroma complexity. Production kinetics and final content of several volatile compounds are also differentially influenced by standard or excess ammonium supplementation.

Metabolic flux analysis during the exponential growth phase of Saccharomyces cerevisiae in wine fermentations.

As a consequence of the increase in global average temperature, grapes with the adequate phenolic and aromatic maturity tend to be overripe by the time of harvest, resulting in increased sugar concentrations and imbalanced C/N ratios in fermenting musts. This fact sets obvious additional hurdles in the challenge of obtaining wines with reduced alcohols levels, a new trend in consumer demands. It would therefore be interesting to understand Saccharomyces cerevisiae physiology during the fermentation of must with these altered characteristics. The present study aims to determine the distribution of metabolic fluxes during the yeast exponential growth phase, when both carbon and nitrogen sources are in excess, using continuous cultures. Two different sugar concentrations were studied under two different winemaking temperature conditions. Although consumption and production rates for key metabolites were severely affected by the different experimental conditions studied, the general distribution of fluxes in central carbon metabolism was basically conserved in all cases. It was also observed that temperature and sugar concentration exerted a higher effect on the pentose phosphate pathway and glycerol formation than on glycolysis and ethanol production. Additionally, nitrogen uptake, both quantitatively and qualitatively, was strongly influenced by environmental conditions. This work provides the most complete stoichiometric model used for Metabolic Flux Analysis of S. cerevisiae in wine fermentations employed so far, including the synthesis and release of relevant aroma compounds and could be used in the design of optimal nitrogen supplementation of wine fermentations.