Jean-Louis Roustan

Modulation of Glycerol and Ethanol Yields During Alcoholic Fermentation in Saccharomyces cerevisiae Strains Overexpressed or Disrupted for GPD1 Encoding Glycerol 3-Phosphate Dehydrogenase

The possibility of the diversion of carbon flux from ethanol towards glycerol in Saccharomyces cerevisiae during alcoholic fermentation was investigated. Variations in the glycerol 3‐phosphate dehydrogenase (GPDH) level and similar trends for alcohol dehydrogenase (ADH), pyruvate decarboxylase and glycerol‐3‐phosphatase were found when low and high glycerol‐forming wine yeast strains were compared. GPDH is thus a limiting enzyme for glycerol production. Wine yeast strains with modulated GPD1 (encoding one of the two GPDH isoenzymes) expression were constructed and characterized during fermentation on glucose‐rich medium. Engineered strains fermented glucose with a strongly modified [glycerol] : [ethanol] ratio. gpd1Δ mutants exhibited a 50% decrease in glycerol production and increased ethanol yield. Overexpression of GPD1 on synthetic must (200 g/l glucose) resulted in a substantial increase in glycerol production (×4) at the expense of ethanol. Acetaldehyde accumulated through the competitive regeneration of NADH via GPDH. Accumulation of by‐products such as pyruvate, acetate, acetoin, 2,3 butane‐diol and succinate was observed, with a marked increase in acetoin production.

Effectiveness of combined ammoniacal nitrogen and oxygen additions for completion of sluggish and stuck wine fermentations

Abstract The timing of combined additions of oxygen and assimilable nitrogen was optimized. Five mg/l oxygen and 300 mg/l (NH4)2HPO4 were added (i) before inoculation, (ii) at the end of the cell growth phase or (iii) at the halfway point of fermentation. There were marked differences between the nine combinations. In all cases, adding oxygen at the end of cell growth and nitrogen at the halfway point of fermentation appeared to be the most effective combination for completion of the fermentation whereas initial additions always had little favorable effect on this completion. The poor effect of initial oxygenation was partially explained by the large fraction of antimycin-sensitive oxygen consumption at the beginning of fermentation. The technological usefulness of optimized combined additions was shown by testing them during 10 different sluggish fermentations: the mean duration was almost 50% of the mean duration for control fermentations.

Nitrogen demand of different yeast strains during alcoholic fermentation. Importance of the stationary phase

The nitrogen demand of industrial yeast strains were compared. Substantial differences were found between strains. These did not change regardless of the initial medium composition and added nitrogen source. To separately study growth and stationary phases, we ran fermentations with different nitrogen feeding profiles: a) exponentially fed fermentations with a long growth phase, and b) constant rate fermentations with nitrogen addition during the stationary phase. Differences between stains mostly appeared during the second phase. Measuring nitrogen requirements under such conditions would thus be an interesting complementary test when selecting new strains especially for enological purposes since most fermentation kinetics are nitrogen limited.

Use of constant rate alcoholic fermentations to compare the effectiveness of different nitrogen sources added during the stationary phase

Alcoholic fermentations by Saccharomyces cerevisiae were run at a constant rate throughout most of the fermentation process by controlling the addition of assimilable nitrogen. During the regulation period, the nitrogen content of the cells and their specific activities were kept constant. Such fermentations were used to compare the effectiveness of additions of different amino acids during the stationary phase. Marked differences were found as compared to fermentations with additions in the initial medium.

Impact of the addition of electron acceptors on the by-products of alcoholic fermentation

Abstract In this study, we investigated the consequences of adding electron acceptors on the production of compounds involved in the maintenance of redox equilibrium in yeast during alcoholic fermentation: glycerol, succinate, acetate, malate, acetoin and butanediol. The various mechanisms involved are only fully functional in the stationary phase. They demonstrate in particular: (i) that in these conditions, the synthesis of glycerol serves more as a means of eliminating a surplus of reducing power than as a means of protection against osmotic pressure; (ii) the importance of the synthesis of acyloins, such as acetoin, followed by reduction to diols. The results obtained also provide information concerning the synthetic pathways for succinate and acetate. These reactions are important in the production of alcoholic drinks (particularly in wine production), because they concern compounds that affect the organoleptic qualities of the products. They may also be of value for the stereospecific synthesis of certain diol-like compounds.

Trehalose and glycogen in wine-making yeasts: methodological aspects and variability

Trehalose and glycogen, which can represent up to 30% of wine yeasts, was evaluated by different methods in (i) yeasts during fermentation of musts (200 g sugar l−1) and (ii) active dry yeasts. Fermentation trials demonstrated the potential value of monitoring changes in trehalose concentration during the rehydration step so that the performance of the yeasts can be evaluated.

Feasibility of measuring ferricyanide reduction by yeasts to estimate their activity during alcoholic fermentation in wine-making conditions.

We assessed the feasibility of measuring the extracellular reduction of ferricyanide in the presence of an intermediate carrier (menadione) as a means of estimating the activity of yeasts during alcoholic fermentation. A spectrophotometric and a potentiometric approach were used. Comparison of specific reductase activity and gas production rate during the stationary phase indicated that measuring the menadione-catalyzed reduction of ferricyanide provides a good estimate of the total activity of the yeast cells in a fermenting must. The response observed following the addition of an electron acceptor (acetaldehyde) confirmed that the reductase activity of menadione is dependent on the availability of NADH. The stability of menadione in the fermentation medium, as assessed by the potentiometric method, suggested that electrochemical reoxidation of the ferrocyanide can act as a substitute for the addition of an electron acceptor when studying the redox regulation of fermenting yeasts.

Role of trehalose and glycogen in alcoholic fermentation in wine-making conditions

Abstract This is a study of the roles of trehalose and glycogen during alcoholic fermentation in wine-making conditions, which is a highly specific and unfavourable environment. We monitored changes in the concentrations of these compounds during fermentation and then studied the effects of the main fermentation parameters and compared the responses to those obtained in classically studied stress conditions. Despite the difficulties in dissociating the various factors thought to affect the synthesis of glycogen and trehalose, this work provides new information about (1) the role of both compounds as storage substances and (2) the additional role of trehalose as a ‘compatible solute’ and as a ‘stress response factor’ in wine-making fermentations.