Untargeted Metabolomics Approach Using UHPLC-HRMS to Unravel the Impact of Fermentation on Color and Phenolic Composition of Rosé Wines.

Color is a major quality trait of rosé wines due to their packaging in clear glass bottles. This color is due to the presence of phenolic pigments extracted from grapes to wines and products of reactions taking place during the wine-making process. This study focuses on changes occurring during the alcoholic fermentation of Syrah, Grenache and Cinsault musts, which were conducted at laboratory (250 mL) and pilot (100 L) scales. The color and phenolic composition of the musts and wines were analyzed using UV-visible spectrophotometry, and metabolomics fingerprints were acquired by ultra-high performance liquid chromatography-high-resolution mass spectrometry. Untargeted metabolomics data highlighted markers of fermentation stage (must or wine) and markers related to the grape variety (e.g., anthocyanins in Syrah, hydroxycinnamates and tryptophan derivatives in Grenache, norisoprenoids released during fermentation in Cinsault). Cinsault wines contained higher molecular weight compounds possibly resulting from the oxidation of phenolics, which may contribute to their high absorbance values.

Unravelling copper effect on the production of varietal thiols during Colombard and Gros Manseng grape juices fermentation by Saccharomyces cerevisiae.

Nowadays the rapidly increasing organic vineyard management with the utilization of copper as sole fungal control pesticide against downy mildew raises once again the question of copper impact on varietal thiols in wine. For this purpose, Colombard and Gros Manseng grape juices were fermented under different copper levels (from 0.2 to 3.88 mg/l) to mimic the consequences in must of organic practices. The consumption of thiol precursors and the release of varietal thiols (both free and oxidized forms of 3-sulfanylhexanol and 3-sulfanylhexyl acetate) were monitored by LC-MS/MS. It was found that the highest copper level (3.6 and 3.88 mg/l for Colombard and Gros Manseng respectively) significantly increased yeast consumption of precursors (by 9.0 and 7.6% for Colombard and Gros Manseng respectively). For both grape varieties, free thiol content in wine significantly decreased (by 84 and 47% for Colombard and Gros Manseng respectively) with the increase of copper in the starting must as already described in the literature. However, the total thiol content produced throughout fermentation was constant regardless of copper conditions for the Colombard must, meaning that the effect of copper was only oxidative for this variety. Meanwhile, in Gros Manseng fermentation, the total thiol content increased along with copper content, resulting in an increase up to 90%; this suggests that copper may modify the regulation of the production pathways of varietal thiols, also underlining the key role of oxidation. These results complement our knowledge on copper effect during thiol-oriented fermentation and the importance of considering the total thiol production (reduced+oxidized) to better understand the effect of studied parameters and differenciate chemical from biological effects.

Modelling the effects of assimilable nitrogen addition on fermentation in oenological conditions.

Alcoholic fermentation in oenological conditions is a biological process carried out under significant physiological constraints: deficiency of nitrogen and other nutriments (vitamins, lipids …) and different stresses (pH and osmotic). In literature, few models have been proposed to describe oenological fermentations. They focused on the initial conditions and did not integrate nitrogen addition during the fermentation process which is a widespread practice. In this work, two dynamic models of oenological fermentation are proposed to predict the effects of nitrogen addition at two different timings: at the beginning of the process and during the fermentation experiment. They were validated and compared against existing models showing an accurate fit to experimental data for CO2 release and CO2 production rate.

Analysis of volatile compounds production kinetics: A study of the impact of nitrogen addition and temperature during alcoholic fermentation.

Among the different compounds present in the must, nitrogen is an essential nutrient for the management of fermentation kinetics, also playing a major role in the synthesis of fermentative aromas. Fermentation temperature is yet another variable that affects fermentation duration and the production of fermentative aromas in wine. The main objective of this study was thus to evaluate the combined effects of nitrogen addition-at the start of the fermentation process or during the stationary phase-at different fermentation temperatures on both fermentation kinetics and aroma synthesis kinetics. To study the impact of these three parameters simultaneously, we used an innovative transdisciplinary approach associating an online GC-MS system with an original modeling approach: a Box-Behnken experimental design combined with response surface modeling and GAM modeling. Our results indicated that all three factors studied had significant effects on fermentation and aroma production kinetics. These parameters did not impact in the same way the different families of volatile compounds. At first, obtained data showed that reduction of ester accumulation in the liquid phase at high temperature was mainly due to important losses by evaporation but also to modifications of yeast metabolic capabilities to synthetize these compounds. In a noticeable way, optimal temperature changed for liquid accumulation of the two classes of esters-23°C for acetate ester and 18°C for ethyl esters-because biological impact of temperature was different for the two chemical families. Moreover, the study of these three factors simultaneously allowed us to show that propanol is not only a marker of the presence of assimilable nitrogen in the medium but above all a marker of cellular activity. Finally, this work enabled us to gain a deeper understanding of yeast metabolism regulation. It also underlines the possibility to refine the organoleptic profile of a wine by targeting the ideal combination of fermentation temperature with initial and added nitrogen concentrations. Such observation was particularly true for isoamyl acetate for which interactions between the three factors were very strong.

New Light on the Varietal Thiols Pathway during Alcoholic Fermentation: Role of 3-S-(N-Acetyl-cysteinyl)-hexan-1-ol (NAC3SH).

For many years, knowledge on thiol precursors has been limited to S-conjugates of glutathione (G3SH), cysteine (Cys3SH), and later on the dipeptides γ-GluCys and CysGly. In this work, we took the parallel between precursor degradation and the glutathione-mediated detoxification pathway a step further by considering a new type of derivative, 3-S-(N-acetyl-l-cysteinyl)hexanol (NAC3SH). This compound was synthesized and then added to the existing liquid chromatography with tandem mass spectrometry (LC-MS/MS) method of thiol precursors. This intermediate was only identified during alcoholic fermentation in synthetic must spiked with G3SH (1 mg/L or 2.45 µmol/L) in the presence of copper with concentration above 1.25 mg/L, which demonstrates for the first time the existence of this new derivative (until 126 µg/L or 0.48 µmol/L) and the capacity of the yeast to produce such a compound. Its status as a precursor was also studied during fermentation, in which a release of 3-sulfanylhexanol was noted corresponding to a conversion yield close to 0.6%. This work completed the thiol precursor's degradation pathway in Saccharomyces cerevisiae in synthetic conditions with a new intermediate, confirming its connection with the xenobiotic detoxification pathway and giving new insights on the precursor's fate.

The aromatic profile of wine distillates from Ugni blanc grape musts is influenced by the nitrogen nutrition (organic vs. inorganic) of Saccharomyces cerevisiae.

Although the impact of nitrogen nutrition on the production of fermentative aromas in oenological fermentation is well known today, one may wonder whether the effects studied are the same when winemaking takes place at high turbidities, specifically for the production of wines intended for cognac distillation. To that effect, a fermentation robot was used to analyze 30 different fermentation conditions at two turbidity levels with several factors tested: (i) initial addition of nitrogen either organic (with a mixture of amino acids - MixAA) or inorganic with di-ammonium phosphate (DAP) at different concentrations, (ii) variation of the ratio of inorganic/organic nitrogen (MixAA and DAP) and (iii) addition of different single amino acids (alanine, arginine, aspartic acid and glutamic acid). A metabolomic analysis was carried out on all resulting wines to have a global vision of the impact of nitrogen on more than sixty aromatic molecules of various families. Then, at the end of the alcoholic fermentation, the wines were micro-distilled. A first interesting observation was that the aroma profiles of both wines and distillates were close, indicating that the concentration factor is rather similar for the different aromas studied. Secondly, the fermentation kinetics and aroma results have shown that the nitrogen concentration effect prevailed over the nature of nitrogen. Although the lipid concentration was in excess, an interaction between the assimilable nitrogen and lipid contents was still observed in wines or in micro-distillates. Alanine is involved in the synthesis of acetaldehyde, isobutanol, isoamyl alcohol and isoamyl acetate. Finally, it was demonstrated that modifying the ratio of assimilable nitrogen in musts is not an interesting technological response to improve the aromatic profile of wines and brandies. Indeed, unbalance the physiological ratio of the must by adding a single source of assimilable nitrogen (organic or inorganic) has been shown to deregulate the synthesis of most of the fermentation aromas produced by the yeast. Wine metabolomic analysis confirmed the results that had been observed in micro-distillates but also in the other aromatic families, especially on terpenes. The contribution of solid particles, but also yeast biosynthesis (via sterol management in must) to wine terpenes is discussed. Indeed, the synthesis of terpenes in this oenological context seems to be favored, especially since the concentration of assimilable nitrogen (in addition to the lipid content) favor their accumulation in the medium. A non-negligible vintage effect on the terpene profile was also demonstrated with variations in their distribution depending on the years. Thus, the present study focuses on the metabolism of wine yeasts under different environmental conditions (nitrogen and lipid content) and on the impact of distillation on the fate of flavor compounds. The results highlight once again the complexity of metabolic fluxes and of the impact of nitrogen source (nature and amount) and of lipids. Furthermore, this study demonstrates that beyond the varietal origin of terpenes, the part resulting from the de novo synthesis by the yeast during the fermentation cannot be neglected in the context of cognac winemaking with high levels of turbidity.

Multi-method study of the impact of fermentation on the polyphenol composition and color of Grenache, Cinsault, and Syrah rosé wines.

Rosé wines show large color diversity, due to different phenolic pigment compositions. However, the mechanisms responsible for such diversity are poorly understood. The present work aimed at investigating the impact of fermentation on the color and composition of rosé wines made from Grenache, Cinsault, and Syrah grapes. Targeted MS analysis showed large varietal differences in must and wine compositions, with higher concentrations of anthocyanins and flavanols in Syrah. UV-visible spectrophotometry and size exclusion chromatography data indicated that Grenache and Cinsault musts contained oligomeric pigments derived from hydroxycinnamic acids and flavanols which were mostly lost during fermentation due to adsorption on lees. Syrah must color was mainly due to anthocyanins which were partly converted to derived pigments through reactions with yeast metabolites with limited color drop during fermentation. This work highlighted the impact of must composition, reflecting varietal characteristics, on changes occurring during fermentation and consequently wine color.

Influence of ergosterol and phytosterols on wine alcoholic fermentation with Saccharomyces cerevisiae strains.

Sterols are a fraction of the eukaryotic lipidome that is essential for the maintenance of cell membrane integrity and its good functionality. During alcoholic fermentation, they enhance yeast growth, metabolism and viability, as well as resistance to high sugar content and ethanol stress. Grape musts clarified in excess lead to the loss of solid particles rich in sterols, resulting in sluggish and stuck fermentations. Two sterol sources can help Saccharomyces cerevisiae yeasts to adapt to fermentation stress conditions: ergosterol (synthesized by yeast under aerobic conditions) and phytosterols (plant sterols imported by yeast cells from grape musts under anaerobiosis). Little is known about the physiological impact of phytosterols assimilation in comparison with ergosterol and the influence of sterol type on fermentation kinetics parameters. Moreover, studies to date have analyzed a limited number of yeast strains. Thus, the aim of this work was to compare the performances of a set of Saccharomyces cerevisiae wine strains that represent the diversity of industrial wine yeast, fermenting with phytosterols or ergosterol under two conditions: sterol limitation (sterol starvation) and high sugar content (the most common stress during fermentation). Results indicated that yeast cell viability was negatively impacted by both stressful conditions, resulting in sluggish and stuck fermentations. This study revealed the huge phenotype diversity of the S. cerevisiae strains tested, in particular in terms of cell viability. Indeed, strains with better viability maintenance completed fermentation earlier. Interestingly, we showed for the first time that sterol type differently affects a wide variety of phenotype, such as viability, biomass, fermentation kinetics parameters and biosynthesis of carbon central metabolism (CCM) metabolites. Ergosterol allowed preserving more viable cells at the end of fermentation and, as a consequence, a better completion of fermentation in both conditions tested, even if phytosterols also enabled the completion of alcoholic fermentation for almost all strains. These results highlighted the essential role of sterols during wine alcoholic fermentation to ensure yeast growth and avoid sluggish or stuck fermentations. Finally, this study emphasizes the importance of taking into account sterol types available during wine fermentation.

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.

Elucidating the Color of Rosé Wines Using Polyphenol-Targeted Metabolomics.

The color of rosé wines is extremely diverse and a key element in their marketing. It is due to the presence of anthocyanins and of additional pigments derived from them and from other wine constituents. To explore the pigment composition and determine its links with color, 268 commercial rosé wines were analysed. The concentration of 125 polyphenolic compounds was determined by a targeted metabolomics approach using ultra high-performance liquid chromatography coupled to triple quadrupole mass spectrometry (UHPLC-QqQ-MS) analysis in the Multiple Reaction Monitoring (MRM) mode and the color characterised by spectrophotometry and CieLab parameters. Chemometrics analysis of the composition and color data showed that although color intensity is primarily determined by polyphenol extraction (especially anthocyanins and flavanols) from the grapes, different color styles correspond to different pigment compositions. The salmon shade of light rosé wines is mostly due to pyranoanthocyanin pigments, resulting from reactions of anthocyanins with phenolic acids and pyruvic acid, a yeast metabolite. Redness of intermediate color wines is related to anthocyanins and carboxypoyranoanthocyanins and that of dark rosé wines to products of anthocyanin reactions with flavanols while yellowness of these wines is associated to oxidation.

Impact of high lipid contents on the production of fermentative aromas during white wine fermentation.

In Cognac, the musts are rich in grape solids and fermentations are usually run with turbidities ranging between 500 and 1500 NTU (nephelometric turbidity unit). These conditions, considered favourable for generating the desired organoleptic profiles of the final Eaux-de-vies, are unusual in winemaking, and, consequently, their impact on yeast metabolism is poorly understood. This study aims to better describe and understand the synthesis of fermentative aromas in such lipid-excess conditions, while integrating the effect of two other very important parameters: the initial concentration of assimilable nitrogen and the temperature of fermentation. To reach this objective, a Box-Behnken design was implemented to describe and model the simple effects of these factors as well as their interactions. Although the lipid concentration was very high, impacts on the production of fermentative aromas were observed. Indeed, high lipid levels promoted the synthesis of higher alcohols. Observing this effect was surprising because there is no metabolic connection between the anabolic pathways of production of these alcohols and the lipid pathway. This effect may be partly explained by impairment in the activity of alcohol acetyl transferases in the presence of lipids, which catalyse the conversion of higher alcohols into the corresponding esters. Therefore, in this study, the negative impact of turbidity was very significant on acetate esters related to the production of acetyl-CoA, which was the main molecule disturbed by the strong presence of lipids. Finally, and more surprisingly, lipid intake did not impact the synthesis of ethyl esters, which depended on the concentration of exogenous lipids. KEY POINTS: • Innovative work on the fermentation of white wine musts with very high lipid contents. • Precise fermentation management and monitoring in Cognac-making conditions. • Experimental design to study the impact of lipids, assimilable nitrogen and temperature on fermentative aroma synthesis.

Correction: Investigations of the mechanisms of interactions between four non-conventional species with Saccharomyces cerevisiae in oenological conditions.

[This corrects the article DOI: 10.1371/journal.pone.0233285.].

Comprehensive study of the dynamic interaction between SO2 and acetaldehyde during alcoholic fermentation.

In this work, we focused on the effect of the initial content of SO2 in synthetic grape juice on yeast metabolism linked to the production of acetaldehyde. Lengthening of the lag phase duration was observed with an increase in the initial SO2 content. Nevertheless, an interesting finding was a threshold value of an initial SO2 content of 30 mg L-1 in the juice led to equilibrium between intracellular SO2 diffusion and SO2 production from the sulfate pool by yeast. The ratios of free and bound acetaldehydes were measured during fermentation, and the maximum accumulation of free acetaldehyde was observed when SO2 concentration equilibrium between diffusion and production was reached in the fermenting juice. Moreover, it was observed that SO2 addition resulted in significant changes in the synthesis of aroma compounds. Production of volatile molecules related to sulfur metabolism (methionol) was changed. But, more surprisingly, synthesis of some volatile carbon compounds (diacetyl, isoamyl alcohol, isobutyl alcohol, phenyl ethanol and their corresponding esters) was also altered because of major disruptions in the NADPH/NADP+ redox equilibrium. Finally, we demonstrated that acetaldehyde bound to SO2 could not be metabolized by the yeast during the time course of fermentation and that only free acetaldehyde could impact metabolism.

Investigations of the mechanisms of interactions between four non-conventional species with Saccharomyces cerevisiae in oenological conditions.

Fermentation by microorganisms is a key step in the production of traditional food products such as bread, cheese, beer and wine. In these fermentative ecosystems, microorganisms interact in various ways, namely competition, predation, commensalism and mutualism. Traditional wine fermentation is a complex microbial process performed by Saccharomyces and non-Saccharomyces (NS) yeast species. To better understand the different interactions occurring within wine fermentation, isolated yeast cultures were compared with mixed co-cultures of one reference strain of S. cerevisiae with one strain of four NS yeast species (Metschnikowia pulcherrima, M. fructicola, Hanseniaspora opuntiae and H. uvarum). In each case, we studied population dynamics, resource consumed and metabolites produced from central carbon metabolism. This phenotyping of competition kinetics allowed us to confirm the main mechanisms of interaction between strains of four NS species. S. cerevisiae competed with H. uvarum and H. opuntiae for resources although both Hanseniaspora species were characterized by a strong mortality either in mono or mixed fermentations. M. pulcherrima and M. fructicola displayed a negative interaction with the S. cerevisiae strain tested, with a decrease in viability in co-culture. Overall, this work highlights the importance of measuring specific cell populations in mixed cultures and their metabolite kinetics to understand yeast-yeast interactions. These results are a first step towards ecological engineering and the rational design of optimal multi-species starter consortia using modeling tools. In particular the originality of this paper is for the first times to highlight the joint-effect of different species population dynamics on glycerol production and also to discuss on the putative role of lipid uptake on the limitation of some non-conventional species growth although interaction processes.

Impact of the timing and the nature of nitrogen additions on the production kinetics of fermentative aromas by Saccharomyces cerevisiae during winemaking fermentation in synthetic media.

During alcoholic fermentation, many parameters, including the nitrogen composition of the must, can affect aroma production. The aim of this study was to examine the impact of several types of nitrogen sources added at different times during fermentation. Nitrogen was added as ammonium or a mixture of amino acids at the beginning of fermentation or at the start of the stationary phase. These conditions were tested with two Saccharomyces cerevisiae strains that have different nitrogen requirements. The additions systematically reduced the fermentation duration. The aroma production was impacted by both the timing of the addition and the composition of the nitrogen source. Propanol appeared to be a metabolic marker of the presence of assimilable nitrogen in the must. The production of ethyl esters was slightly higher after the addition of any type of nitrogen; the production of higher alcohols other than propanol was unchanged, and acetate esters were overproduced due to the overexpression of the genes ATF1 and ATF2. Finally the parameter affecting the most the synthesis of beneficial aromas was the addition timing: The supply of organic nitrogen at the beginning of the stationary phase was more favorable for the synthesis of beneficial aromas.

Comprehensive Study of the Evolution of the Gas-Liquid Partitioning of Acetaldehyde during Wine Alcoholic Fermentation.

Determining the gas-liquid partitioning ( Ki) of acetaldehyde during alcoholic fermentation is an important step in the optimization of fermentation control with the aim of minimizing the accumulation of this compound, which is responsible for the undesired attributes of green apples and fresh-cut grass in wines. In this work, the effects of the main fermentation parameters on the Ki of acetaldehyde were assessed. Ki values were found to be dependent on the temperature and composition of the medium. A nonlinear correlation between the evolution of the Ki and fermentation progress was observed, attributable to the strong retention effect of ethanol at low concentrations, and it was demonstrated that the partitioning of this specific molecule was not influenced by the CO2 production rate. A model was developed that quantifies the Ki of acetaldehyde with a very accurate prediction, as the difference between the observed and predicted values did not exceed 9%.

Vicinal diketones and their precursors in wine alcoholic fermentation: Quantification and dynamics of production.

Vicinal diketones produced during wine fermentation influence the organoleptic qualities of wine. Diacetyl and 2,3-pentanedione are well known for their contribution to butter or butterscotch-like flavours. We developed an analysis method to quantify vicinal diketones and their precursors, α-acetolactate and α-acetohydroxybutyrate, under oenological conditions. Five-fold dilution of the sample in a phosphate-citrate buffer (pH7.0) strongly attenuated matrix effects between the beginning and end of alcoholic fermentation and protected the sample from spontaneous precursor decarboxylation. The use of diacetyl-d6 as an internal reference improved precision by eliminating differences in the derivatization and extraction yields between the internal standard and the analytes. We obtained unexpected results for alcoholic fermentation by Saccharomyces cerevisiae using this approach. Indeed, the level of diacetyl and 2,3-pentanedione throughout fermentation were very low. However, we observed a large quantity of both precursors. The production dynamics of α-acetolactate were unconventional and there were two distinct phases of accumulation. The first corresponded to the growth phase, and the second to glucose depletion. There was a rapid decrease of precursor levels at the end of fermentation, but there was still a significant amount of α-acetolactate. The amount of precursor remaining at the end of fermentation constitutes a potential source of diacetyl during wine maturation. α-Acetohydroxybutyrate accumulated during the growth phase followed by a continuous decrease of its concentration during the stationary phase. Residual quantities of α-acetohydroxybutyrate found in wine at the end of fermentation does not constitute a sufficient source of 2,3-pentanedione to affect the aromatic profile.

Quantitative 13 C-isotope labelling-based analysis to elucidate the influence of environmental parameters on the production of fermentative aromas during wine fermentation.

Nitrogen and lipids are key nutrients of grape must that influence the production of fermentative aromas by wine yeast, and we have previously shown that a strong interaction exists between these two nutrients. However, more than 90% of the acids and higher alcohols (and their acetate ester derivatives) were derived from intermediates produced by the carbon central metabolism (CCM). The objective of this study was to determine how variations in nitrogen and lipid resources can modulate the contribution of nitrogen and carbon metabolisms for the production of fermentative aromas. A quantitative analysis of metabolism using 13 C-labelled leucine and valine showed that nitrogen availability affected the part of the catabolism of N-containing compounds, the formation of α-ketoacids from CCM and the redistribution of fluxes around these precursors, explaining the optimum production of higher alcohols occurring at an intermediate nitrogen content. Moreover, nitrogen content modulated the total production of acids and higher alcohols differently, through variations in the redox state of cells. We also demonstrated that the phytosterol content, modifying the intracellular availability of acetyl-CoA, can influence the flux distribution, especially the formation of higher alcohols and the conversion of α-ketoisovalerate to α-ketoisocaproate.

Impact of initial lipid content and oxygen supply on alcoholic fermentation in champagne-like musts.

Available nitrogen, lipids, or oxygen are nutrients with major impact on the kinetics of winemaking fermentation. Assimilable nitrogen is usually the growth-limiting nutrient which availability determines the fermentation rate and therefore the fermentation duration. In some particular cases, as in Champagne, grape musts have high available nitrogen content and low turbidity, i.e., below 50 Nephelometric Turbidity Unit (NTU). In the case of low turbidity, the availability of lipids, particularly phytosterols, becomes limiting. In this situation, control of oxygenation, which is necessary for lipid synthesis by yeast, is particularly crucial during fermentation. To mimic and understand these situations, a synthetic medium simulating the average composition of a Champagne must was used. This medium contained phytosterol (mainly ß-sitosterol) concentrations ranging from 0 to 8mg/L corresponding to turbidity between 10 and 90 NTU. Population reached during the stationary phase and the maximum fermentation rate are conditioned by the initial phytosterol concentration determining the amount of nitrogen consumption. An early loss of viability was observed when the lipid concentrations were very low. For example, the viability continuously decreased during the stationary phase to a final value of 50% for an initial phytosterol concentration of 1mg/L. In some fermentations, 10mg/L oxygen were added at the end of the growth phase to combine the effects of initial content of phytosterols in the musts and the de novo synthesis of ergosterol and unsaturated fatty acids induced by oxygen addition. Effect of oxygen supply on the fermentation kinetics was particularly significant for media with low phytosterol contents. For example, the maximum fermentation rate was increased by 1.4-fold and the fermentation time was 70h shorter with oxygen addition in the medium containing 2mg/L of phytosterols. As a consequence of the oxygen supply, for the media containing 3, 5 and 8mg/L of phytosterols, the assimilable nitrogen was completely exhausted and the fermentation kinetics, as well as the final populations and viabilities (greater than 90%), were identical for the 3 conditions. The impacts of the lipid content and additional oxygen on acetate, glycerol and succinate synthesis were also studied. The phytosterols decreased the acetate and increased the succinate synthesis, and oxygenation resulted in a decrease in succinate formation. This work highlights the similarities and differences between the effects of lipids and oxygen on fermentation kinetics and yeast metabolism. This research highlights the need for an optimal combined management of lipid content in the must via turbidity and oxygenation, particularly in nitrogen-rich musts.