Dynamics and quantitative analysis of the synthesis of fermentative aromas by an evolved wine strain of Saccharomyces cerevisiae.

We performed a dynamic and quantitative analysis of the synthesis of fermentative aromas by an aromatic wine yeast, ECA5, obtained by adaptive evolution. During fermentation at pilot scale on synthetic and natural musts, ECA5 produced volatile compounds (higher alcohols and their acetates, ethyl esters) at higher rates than the ancestral strain, with the exception of propanol. Marked differences in the chronology of synthesis of several compounds were observed between the two strains. Overproduction of phenyl ethanol occurred mainly during the growth phase for ECA5, consistent with its higher flux through the pentose phosphate pathway, which plays a key role in biosynthetic processes. The kinetics of production of isobutanol and isoamyl alcohol were differently affected by different media (synthetic or natural must) and, in particular, according to the nature of the sterols in the media (ergosterol or phytosterols). We also observed differences in the chronology of synthesis of ethyl acetate and isoamyl acetate or ethyl esters, suggesting that the regulation of the synthesis of these compounds in the evolved strain differs from that in the ancestral strain. This study shows that a dynamic analysis of volatile compounds, using high acquisition frequency online gas chromatography, can provide novel insights into the synthesis and regulation of aromas and is thus a potentially powerful tool for strain characterization.

Nitrogen-backboned modeling of wine-making in standard and nitrogen-added fermentations.

Nitrogen has a strong impact on the key bio-mechanisms involved during the grape-must fermentation but also on the synthesis of flavour markers determining the aromatic profile of the wine. This paper first presents a consistent dynamical mass balance model describing the main physiological phenomena implied in standard batch fermentations, i.e. consumption of sugar and nitrogen and synthesis of ethanol. It also includes nitrogen compounds such as hexose transporters. Moreover, a common practice in wine-making is the addition of nitrogen during the fermentation in order to boost and shorten the process duration. A tractable representation of this boost effect has therefore been developed as an extension of the first model. It is apparent that yeast makes a different use of nitrogen depending on the fermentation stage at which the addition is effected, balancing the regrowth of biomass and the synthesis of supplementary hexose transporters. These models have been validated in line with experimental evidence deduced from extensive experimental studies.

Metabolic responses of Saccharomyces cerevisiae to valine and ammonium pulses during four-stage continuous wine fermentations.

Nitrogen supplementation, which is widely used in winemaking to improve fermentation kinetics, also affects the products of fermentation, including volatile compounds. However, the mechanisms underlying the metabolic response of yeast to nitrogen additions remain unclear. We studied the consequences for Saccharomyces cerevisiae metabolism of valine and ammonium pulses during the stationary phase of four-stage continuous fermentation (FSCF). This culture technique provides cells at steady state similar to that of the stationary phase of batch wine fermentation. Thus, the FSCF device is an appropriate and reliable tool for individual analysis of the metabolic rerouting associated with nutrient additions, in isolation from the continuous evolution of the environment in batch processes. Nitrogen additions, irrespective of the nitrogen-containing compound added, substantially modified the formation of fermentation metabolites, including glycerol, succinate, isoamyl alcohol, propanol, and ethyl esters. This flux redistribution, fulfilling the requirements for precursors of amino acids, was consistent with increased protein synthesis resulting from increased nitrogen availability. Valine pulses, less efficient than ammonium addition in increasing the fermentation rate, were followed by a massive conversion of this amino acid in isobutanol and isobutyl acetate through the Ehrlich pathway. However, additional routes were involved in valine assimilation when added in stationary phase. Overall, we found that particular metabolic changes may be triggered according to the nature of the amino acid supplied, in addition to the common response. Both these shared and specific modifications should be considered when designing strategies to modulate the production of volatile compounds, a current challenge for winemakers.

Stuck and slow fermentations in enology: statistical study of causes and effectiveness of combined additions of oxygen and diammonium phosphate.

One hundred and seventy-eight musts from different regions in France were selected by enologists and fermented under standardized conditions. Two kinds of fermentation problems were distinguished: (a) slow and (b) sluggish (late-onset sluggish) and stuck (with residual sugar) fermentations. Slow fermentations, characterized by a low fermentation rate throughout the process, were always due to low assimilable nitrogen concentrations in the must. The advantages of using formol titration for measuring assimilable nitrogen are discussed. In contrast, sluggish and stuck fermentations, characterized by very low yeast viability at the end of fermentation, could not be predicted from the analytical data even though their probability was increased when the initial sugar concentration was high. All problems of stuck and sluggish fermentations (concerning 40% of the musts) were solved by supplying 7 mg/l oxygen and 300 mg/l diammonium phosphate at the halfway of the fermentation process, which confirmed (i) the importance of these two nutrients in enological practices and (ii) the importance of adding them at the right time.

Use of a continuous multistage bioreactor to mimic winemaking fermentation.

Continuous fermentation set-ups are of great interest for studying the physiology of microorganisms. In winemaking conditions, yeasts go through a growth phase and a stationary phase during which more than half of the sugar is fermented. A comprehensive study of wine-yeast physiology must therefore include yeasts in a non-growing phase. This condition is impossible to achieve within a chemostat, which led us to design a multi-stage fermentation device. In this study, we evaluated the ability of such a device to reproduce, in a series of steady states, the conditions of batch fermentation. Two-stage and four-stage fermentations were carried out with two different strains of Saccharomyces cerevisiae. The main characteristics of the fermentation process (biomass growth, by-product content of the medium) were compared with those observed in batch mode at the same stage of fermentation, which was defined by glucose uptake. The four-stage configuration showed a better ability to reproduce batch fermentation characteristics than the two-stage set-up. It also allowed to uncouple the variations of environmental parameters and proved to be a promising tool to gain new insights into yeast metabolism during alcoholic fermentation.

Butanediol production by Aerobacter aerogenes NRRL B199: effects of initial substrate concentration and aeration agitation.

Butanediol production by Aerobacter aerogenes NRRL B199 grown on glucose requires an optimal rate of aeration for the obtention of butanediol 2, 3. In the absence of air, Aerobacter aerogenes NRRL B199 growth and production are weak. Agitation-aeration is necessary for producing the biomass, but an excess of oxygen proves to be toxic with regard to metabolite production. Oxygen is a limiting substrate with regard to growth and an inhibitor with regard to the specific metabolite productivity. This observation is discussed from a kinetic stand point and in relation to the search for the optimum oxygen transfer coefficient (K(L)a), which is found to be in the range of 50-100h(-1). It has also been observed that K(L)a increases during the fermentation cycle. The initial substrate concentration effects the yield production of biomass and butanediol production. Low yields of butanediol are obtained at low initial sugar concentrations, but good yields of butanediol are obtained (0.45 g/g) at high concentrations of glucose (195 g/L). Carbon substrates and butanediol are inhibitors of cell growth while butanediol is not quite an inhibitor of the specific rate of butanediol production for the range of butanediol of 0-100 g/L.

Modeling the effects of assimilable nitrogen and temperature on fermentation kinetics in enological conditions.

We propose a dynamic model of alcoholic fermentation in wine-making conditions. In this model, the speed at which CO(2) is released is related to the effects of the main factors involved in fermentation in wine-making conditions: temperature (which can vary within a predefined range) and nitrogen additions (which must not exceed the maximal authorized level). The resulting model consists of ordinary differential equations including numerous parameters that need to be identified and important interactions between explicative variables. These parameters were identified by uncoupling the effects of variables during specific experiments. The results were validated on another series of experiments in different conditions.

Re-assessment of the influence of yeast strain and environmental factors on glycerol production in wine.

Increasing glycerol production is of concern for wine-makers in improving the quality of certain wines. We have compared the impact of strain and relevant environmental factors influencing glycerol production under the same conditions, i.e. standardized conditions simulating enological fermentation. The glycerol production of 19 industrial wine strains ranged from 6.4 to 8.9 g l-1 and varied significantly between strains. The production of acetate and succinate was also found to differ substantially depending on the strain but no significant strain-dependent variation was observed for acetaldehyde. Interestingly, high glycerol production was not correlated to high production of acetate or acetaldehyde, which are undesirable in wine. A detailed study with two low or two high glycerol-producing strains showed that temperature and the initial concentration of nitrogen had little effect on the amount of glycerol formed, although agitation or a nitrogen source composed mainly of ammoniacal nitrogen slightly enhanced glycerol production. The influence of environmental factors remained minor while the predominant factor for glycerol variability in wine was attributed to the strain. Taking into account wine-making constraints, the results indicate that achieving a high glycerol content in wine requires the selection or improvement of yeast strains rather than the control of growth and cultivation conditions.