Impact of nutrient imbalance on wine alcoholic fermentations: nitrogen excess enhances yeast cell death in lipid-limited must

Abstract

We evaluated the consequences of nutritional imbalances, particularly lipid/nitrogen imbalances, on wine yeast survival during alcoholic fermentation. We report that lipid limitation (ergosterol limitation in our model) led to a rapid loss of viability during the stationary phase of fermentation and that the cell death rate is strongly modulated by nitrogen availability and nature. Yeast survival was reduced in the presence of excess nitrogen in lipid-limited fermentations. The rapidly dying yeast cells in fermentations in high nitrogen and lipid-limited conditions displayed a lower storage of the carbohydrates trehalose and glycogen than observed in nitrogen-limited cells. We studied the cell stress response using HSP12 promoter-driven GFP expression as a marker, and found that lipid limitation triggered a weaker stress response than nitrogen limitation. We used a SCH9-deleted strain to assess the involvement of nitrogen signalling pathways in the triggering of cell death. Deletion of SCH9 increased yeast viability in the presence of excess nitrogen, indicating that a signalling pathway acting through Sch9p is involved in this nitrogen-triggered cell death. We also show that various nitrogen sources, but not histidine or proline, provoked cell death. Our various findings indicate that lipid limitation does not elicit a transcriptional programme that leads to a stress response protecting yeast cells and that nitrogen excess triggers cell death by modulating this stress response, but not through HSP12. These results reveal a possibly negative role of nitrogen in fermentation, with reported effects referring to ergosterol limitation conditions. These effects should be taken into account in the management of alcoholic fermentations.

Figure 1. Effect of nutrient imbalances during alcoholic fermentation.

Rate of CO₂ production by S. cerevisiae EC1118 cultures at 24°C (A) at various times of fermentation or (B) CO₂ production, and viability of S. cerevisiae EC1118 cells according to (C) time of alcoholic fermentation at 24°C and (D) CO₂ production. The synthetic medium contained 71 mg/L (SM71) or 142 mg/L (SM142) assimilable nitrogen, and 5% or 100% lipid factors (LF5% or LF100%). Viability was measured by flow cytometry after propidium iodide staining. The graphs are the result of smoothing of measurements series (at least 3 repetitions) using the software R. Smoothing obtained is framed by a confidence interval calculated at 95%.

Figure 2. Effect of lipid factors on the viability of S. cerevisiae EC1118 cells during alcoholic fermentation.

The synthetic medium contained 142 mg/L assimilable nitrogen (SM142) and 5% lipid factor (LF5%), with or without additional oleic acid or ergosterol (content as in LF100%). Viability was measured by flow cytometry after propidium iodide staining. The graphs are the result of smoothing of measurements series (at least 3 repetitions) using the software R. Smoothing obtained is framed by a confidence interval calculated at 95%.

Figure 3. Effect of nitrogen source on the viability of S. cerevisiae EC1118 cells during alcoholic fermentation.

The synthetic medium contained 71 mg/L assimilable nitrogen (SM71) and 5% lipid factors (LF5%), with or without additional arginine, glutamine, glutamate, histidine or proline (142 mg/L assimilable nitrogen final content as in SM142). Viability was measured by flow cytometry after propidium iodide staining. The graphs are the result of smoothing of measurements series (at least 3 repetitions) using the software R. Smoothing obtained is framed by a confidence interval calculated at 95%.

Figure 4. Trehalose and glycogen contents at stationary phase of S. cerevisiae EC1118.

Cells were fermented in synthetic medium containing 71 mg/L (SM71) or 142 mg/L (SM142) assimilable nitrogen, and 5% or 100% lipid factors (LF5% or LF100%).

Figure 5. Effect of rapamycin on the viability of S. cerevisiae EC1118 cells during alcoholic fermentation.

The synthetic medium contains 142 mg/L assimilable nitrogen (SM142) and 5% lipid factors (LF5%), with or without 20 nmol/L rapamycin. Viability was measured by flow cytometry after propidium iodide staining. The graphs are the result of smoothing of measurements series (at least 3 repetitions) using the software R. Smoothing obtained is framed by a confidence interval calculated at 95%.

Figure 6. Effect of Sch9 deletion on the cell viability of haploid S. cerevisiae 59A during alcoholic fermentation.

The synthetic medium contains 71 mg/L (SM71) or 142 mg/L (SM142) of assimilable nitrogen, and 5% lipid factor (LF5%). Viability was measured by flow cytometry after propidium iodide staining. The graphs are the result of smoothing of measurements series (at least 3 repetitions) using the software R. Smoothing obtained is framed by a confidence interval calculated at 95%.

Figure 7. Effect of the nutrient imbalances on HSP12 stress gene expression in S. cerevisiae EC1118 during alcoholic fermentation.

The synthetic medium contained 71 mg/L (SM71) or 142 mg/L (SM142) assimilable nitrogen, and 5% or 100% lipid factors (LF5% or LF100%). Fluorescence of the GFP reporter protein, produced under the control of the HSP12 promoter, was measured by flow cytometry. The graphs are the result of smoothing of measurements series (at least 3 repetitions) using the software R. Smoothing obtained is framed by a confidence interval calculated at 95%.