Biomass Content Governs Fermentation Rate in Nitrogen-Deficient Wine Musts

Abstract

Problematic fermentations are common in the wine industry. Assimilable nitrogen deficiency is the most prevalent cause of sluggish fermentations and can reduce fermentation rates significantly. A lack of nitrogen diminishes a yeast's metabolic activity, as well as the biomass yield, although it has not been clear which of these two interdependent factors is more significant in sluggish fermentations. Under winemaking conditions with different initial nitrogen concentrations, metabolic flux analysis was used to isolate the effects. We quantified yeast physiology and identified key metabolic fluxes. We also performed cell concentration experiments to establish how biomass yield affects the fermentation rate. Intracellular analysis showed that trehalose accumulation, which is highly correlated with ethanol production, could be responsible for sustaining cell viability in nitrogen-poor musts independent of the initial assimilable nitrogen content. Other than the higher initial maintenance costs in sluggish fermentations, the main difference between normal and sluggish fermentations was that the metabolic flux distributions in nitrogen-deficient cultures revealed that the specific sugar uptake rate was substantially lower. The results of cell concentration experiments, however, showed that in spite of lower sugar uptake, adding biomass from sluggish cultures not only reduced the time to finish a problematic fermentation but also was less likely to affect the quality of the resulting wine as it did not alter the chemistry of the must.Sluggish and stuck fermentations are common in the wine industry. Factors that affect the yeast growth rate and that lead to problematic fermentations include limited nutrient contents, ethanol toxicity, fatty acid toxicity, temperature extremes (5), the ratio of nitrogen sources to carbon sources in the medium (21), and the initial quantity and quality of the amino acids (29). Sluggish cultures may prolong the process time from days to weeks. In stuck fermentations, high levels of residual sugar are left following the arrest of fermentation. Several difficulties occur at the cellular level; these include diminished sugar transport capacity, the inability of yeasts to produce compatible solutes in response to high osmolality (20,28), and cell membrane integrity issues in the presence of high concentrations of ethanol (8). As such mechanisms may act simultaneously in cells and to various extents, it is difficult to isolate and reproduce the mechanisms in stuck and sluggish fermentations.Quantitatively, nitrogen is the second most abundant nutrient in wine fermentations. It is essential for yeast metabolism and growth. Consequently, a lack of nitrogen triggers sluggish fermentations (1,8,13). In previous studies workers found differences in nitrogen-related curves (e.g., curves of biomass versus assimilable nitrogen) which indicated that the fermentation rate and biomass yield functions are distinct (9, 10; W. Agenbach, Proc. S. Afr. Soc. Enol. Vitic., p. 66-88, 1977). In other studies the ...

Effect of the size of yeast flocs and zinc supplementation on continuous ethanol fermentation performance and metabolic flux distribution under very high concentration conditions

“…However, the amount of DNA was not affected, being constant at the level of 0.5% of the cell dry weight. It was reported that macromolecular compositions of S. cerevisiae were liable to change with the variation of heterologous gene expression (Gonzalez et al, 2003) and environmental conditions such as dilution rate and nitrogen deficiency (Nissen et al, 1997;Varela et al, 2004). Macromolecular compositions such as protein and RNA serve as the basis of metabolic behaviors.…”

“…The metabolic flux analysis of the self-flocculating yeast was performed using the stoichiometric models from Nissen et al (1997) and Varela et al (2004) with modifications that incorporated 10 transport reactions across the membranes, trehalose, and ergosterol synthesis reactions as listed in Appendix.…”

“…Up till now, metabolic profiling and flux analysis have been established for analyzing intermediate metabolites in ethanol fermentation with Saccharomyces cerevisiae under low and normal concentration substrate conditions (Jouhten et al, 2008;Nissen et al, 1997;Varela et al, 2004), but how metabolic flux distribution is affected under VHC conditions has been rarely exploited. Devantier et al (2005) performed metabolic profiling analysis for batch VHC fermentation, but so far no related study has been reported for continuous VHC ethanol fermentation systems.…”

“…Previous reports have documented that the specific growth rate of S. cerevisiae was affected by environmental conditions such as dilution rate, nitrogen deficiency, and glucose repression (Andreas et al, 2001;Nissen et al, 1997;Varela et al, 2004). In our present study, it was found that the size of yeast flocs exerted significant impact on their specific growth rate, and the yeast flocs with larger size showed lower specific growth rate due to potential mass transfer limitation, which should be taken into account in the process optimization to prevent its negative impact on the fermentation performance.…”

Response Surface Optimization of Enzymatic Hydrolysis of Sugar Beet Leaves into Fermentable Sugars for Bioethanol Production

“…The amount of nitrogen required for biomass synthesis can be estimated from the empirical formula for a bacterial cell as C 5 H 7 O 2 N [47]. Previous studies on the effect of nitrogen on ethanol fer-mentation have shown the adverse effects of nitrogen deficiency [48] [49] [50]. The lack of nitrogen diminishes yeast's metabolic activity, sugar uptake rate, and the cell biomass during fermentation.…”