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.

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.

The use of respiration indices in the composting process: a review.

Respiration is directly related to the metabolic activity of a microbial population. Micro-organisms respire at higher rates in the presence of large amounts of bioavailable organic matter while respiration rate is slower if this type of material is scarce. In the composting process respiration activity has become an important parameter for the determination of the stability of compost. It is also used for the monitoring of the composting process and it is considered an important factor for the estimation of the maturity of the material. A wide range of respirometric protocols has been reported based either on CO2 production, O2 uptake or release of heat. The most common methods are those based on O2 uptake. Respirometric assays are affected by a number of parameters including temperature, humidity, and both incubation and pre-incubation conditions. Results from respirometries are generally expressed as 'respiration indices', most of them with their own units and basis. In consequence, some confusion exists when referring and comparing respiration indices. This is particularly important because current and future legislations define and measure the biological stability of waste on the basis of respiration activity of the material. This paper discusses and compares most common respiration indices currently used.

Respirometric assays at fixed and process temperatures to monitor composting process.

A static respirometer was built to determine the respiration index (RI) of composting samples. Respiration indices of different sludges were determined at 37 degrees C (RI37) and at the in situ temperature of the composter at sampling (RI(T)). Results indicated that both indices correlated well with temperature evolution in the composter. RI(T) were more representative of the metabolic activity in the reactor and more sensitive to temperature and composition variations of the composting material, but could not indicate the stability of the material at later stages of the process. Moreover, significance of RI units was shown in the composting of a highly compostable residue. According to the stability limits suggested in the literature, initial RI expressed on dry matter (DM) basis corresponds to a stable material (RI < 1 mgO2gDM(-1)h(-1)) whereas initial RI expressed on organic matter basis (OM) corresponds to an unstable material (RI = 2.5 mgO2gOM(-1)h(-1)).