Impact of nitrogen addition in metabolic flux distribution of Saccharomyces cerevisiae under wine making conditions

Abstract

Metabolic flux analysis (MFA) is a powerful tool for simulating metabolism in scale reconstructions of metabolic networks. This instrument is based on the principle of mass conservation of the intracellular metabolites within a defined stoichiometric network. MFA could be very useful to study the impact of environmental variations on the general metabolism of biological systems, for example, the effect of the variation of nitrogen availability in grape must on yeast during alcoholic fermentations. Saccharomyces cerevisiae is the most relevant yeast species responsible for the alcoholic fermentation that takes place during winemaking. Nitrogen is essential for yeast metabolism and growth. Due to the imbalance between nitrogen and carbon sources available during fermentation of grape juice, the former become a limiting factor. For this reason, assimilable nitrogen is usually measured in wineries and its concentration corrected by the addition of inorganic sources (generally ammonium diphosphate) in order to prevent stuck or sluggish fermentation. However, this supplementation has to be controlled considering that an excess of nitrogen in grape juice could cause the production undesirable aroma compounds by yeast during wine fermentation. Our main goal is to apply metabolic flux analysis to study the effect of nitrogen supplementation on the fermentative metabolism of wine yeast strains. So, different steady states mimicking both an ideal and over addition of nitrogen to must have been achieved using chemostats. A synthetic must with 200 mg/L of yeast assimilable nitrogen (YAN) has been considered as control must. So, the addition of nitrogen to a poor must (100 mg/L of YAN) for achieving 200 mg/L of YAN has been considered as an ideal addition. On the other hand, the addition of 300 mg/L of YAN to the control must has been considered as an over addition of nitrogen. Continuous cultures were run in duplicate at the dilution rate of 0.08 h-1 and at 28ºC in order to simulate the transition from exponential to stationary phase during red wine fermentations. A clear effect of nitrogen over-supplementation on biomass synthesis, CO2 and ethanol production rates, and fusel alcohols production has been observed.