The Timing of Nitrogen Addition Impacts Yeast Genes Expression and the Production of Aroma Compounds During Wine Fermentation.

Among the different compounds present in the must, nitrogen is an essential nutrient for the management of fermentation kinetics but also plays an important role in the synthesis of fermentative aromas. To address the problems related to nitrogen deficiencies, nitrogen additions during alcoholic fermentation have been implemented. The consequences of such additions on the main reaction are well known. However, their impact on aromas synthesis is still poorly understood. So, the main objective of this study was to determine the impact of nitrogen addition during the stationary phase on both the fermentation kinetics and aroma synthesis. To reach this goal, we used a transdisciplinary approach combining statistical modeling (Box-Behnken design and response surface modeling) and gene expression study (transcriptomic analysis). Our results indicated that nitrogen metabolism, central carbon metabolism (CCM), fermentation kinetics and aroma production were significantly impacted by nitrogen addition. The most remarkable point was the different regulation of the bioconversion of higher alcohols into acetate esters on one hand and of fatty acids into ethyl esters on the other hand. We highlighted that the conversion of higher alcohols into acetate esters was maximum when nitrogen was added at the beginning of the stationary phase. Conversely, the highest conversion of acids into ethyl esters was reached when nitrogen was added close to the end of the stationary phase. Moreover, even if the key element in the production of these two ester families appeared to be the enzymatic activity responsible for their production, rather than the availability of the corresponding precursors, these enzymatic activities were differently regulated. For acetate esters, the regulation occurred at gene level: the ATF2 gene was overexpressed following nitrogen addition during the stationary phase. On the opposite, no induction of gene expression was noted for ethyl esters; it seemed that there was an allosteric regulation.

Workflow Based on the Combination of Isotopic Tracer Experiments to Investigate Microbial Metabolism of Multiple Nutrient Sources.

Studies in the field of microbiology rely on the implementation of a wide range of methodologies. In particular, the development of appropriate methods substantially contributes to providing extensive knowledge of the metabolism of microorganisms growing in chemically defined media containing unique nitrogen and carbon sources. In contrast, the management through metabolism of multiple nutrient sources, despite their broad presence in natural or industrial environments, remains virtually unexplored. This situation is mainly due to the lack of suitable methodologies, which hinders investigations. We report an experimental strategy to quantitatively and comprehensively explore how metabolism operates when a nutrient is provided as a mixture of different molecules, i.e., a complex resource. Here, we describe its application for assessing the partitioning of multiple nitrogen sources through the yeast metabolic network. The workflow combines information obtained during stable isotope tracer experiments using selected 13C- or 15N-labeled substrates. It first consists of parallel and reproducible fermentations in the same medium, which includes a mixture of N-containing molecules; however,a selected nitrogen source is labeled each time. A combination of analytical procedures (HPLC, GC-MS) is implemented to assess the labeling patterns of targeted compounds and to quantify the consumption and recovery of substrates in other metabolites. An integrated analysis of the complete dataset provides an overview of the fate of consumed substrates within cells. This approach requires an accurate protocol for the collection of samples-facilitated by a robot-assisted system for online monitoring of fermentations-and the achievement of numerous time-consuming analyses. Despite these constraints, it allowed understanding, for the first time, the partitioning of multiple nitrogen sources throughout the yeast metabolic network. We elucidated the redistribution of nitrogen from more abundant sources toward other N-compounds and determined the metabolic origins of volatile molecules and proteinogenic amino acids.

A comparison of laboratory and pilot-scale fermentations in winemaking conditions.

We investigated the influence of the fermenter size on alcoholic fermentation. Experiments were carried out at pilot scale, in 100-L fermenters, and at laboratory scale, in stirred and static 1-L fermenters. Two musts, Grenache blanc and Sauvignon, were fermented with and without the addition of solid particles from grape musts. Highly clarified must fermentation kinetics was strongly affected by the scale of the experiment, with slower fermentation occurring in the 100-L fermenter. Alcohol, ester, and thiol synthesis in clarified sauvignon must fermentation was also strongly correlated with the fermentation scale. Addition of solid particles from grape tended to reduce the effects on kinetics associated with increasing the scale of the fermentation, by increasing the maximum rate of CO(2) production, and by shortening the duration of fermentation. The addition of such particles also decreased the effects of scaling up the fermentation on the concentration of some volatile compounds, i.e., isoamyl acetate, ethyl octanoate, but did not decrease this effect for other compounds, such as isobutyl acetate, isobutanol, and 3-mercaptohexanol.