Article

Oxygen Response of the Wine Yeast Saccharomyces cerevisiae EC1118 Grown under Carbon-Sufficient, Nitrogen-Limited Enological Condition

Department of Chemical and Bioprocess Engineering, School of Engineering, Pontificia Universidad Católica de Chile, Av. Vicuña Mackenna 4860, Macul, Santiago, Chile.
Applied and Environmental Microbiology (Impact Factor: 3.67). 12/2012; 78(23). DOI: 10.1128/AEM.02305-12

ABSTRACT

Discrete additions of oxygen play a critical role in alcoholic fermentation. However, few studies have quantitated the fate of dissolved oxygen and its impact on wine yeast cell physiology under enological conditions. We simulated the range of dissolved oxygen concentrations that occur after a pump-over during the winemaking process by sparging nitrogen-limited continuous cultures with oxygen-nitrogen gaseous mixtures. When the dissolved oxygen concentration increased from 1.2 to 2.7 μM, yeast cells changed from a fully fermentative to a mixed respirofermentative metabolism. This transition is characterized by a switch in the operation of the tricarboxylic acid cycle (TCA) and an activation of NADH shuttling from the cytosol to mitochondria. Nevertheless, fermentative ethanol production remained the major cytosolic NADH sink under all oxygen conditions, suggesting that the limitation of mitochondrial NADH reoxidation is the major cause of the Crabtree effect. This is reinforced by the induction of several key respiratory genes by oxygen, despite the high sugar concentration, indicating that oxygen overrides glucose repression. Genes associated with other processes, such as proline uptake, cell wall remodeling, and oxidative stress, were also significantly affected by oxygen. The results of this study indicate that respiration is responsible for a substantial part of the oxygen response in yeast cells during alcoholic fermentation. This information will facilitate the development of temporal oxygen addition strategies to optimize yeast performance in industrial fermentations

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    • "These data confirm respiratory metabolism as one major determinant of ethanol yields under these fermentation conditions. The RQ values obtained for S. cerevisiae are in agreement with our previous results (Quir os et al., 2014), as well as other authors, depending on the strain and growth conditions, RQ values ranging from 2.8 to ∞ have been described for S. cerevisiae under aerated glucose rich conditions (Aceituno et al., 2012;de Deken, 1966;Franzen, 2003). It is worth noting that oxygen consumed after 72 h in these cultures ranged from 1.9 g/L to 19.4 g/L (Supplementary file 2), far apart from the microgram or milligram range used in other enological applications . "
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    ABSTRACT: We have recently shown that ethanol yields in winemaking can be reduced by taking advantage of the respiratory metabolism of some non-Saccharomyces yeast species. Using an orthogonal design we have now addressed the impact of three environmental factors (temperature, nitrogen source, and oxygen supply level) on the aerobic metabolism in synthetic must of Saccharomyces cerevisiae, Metschnikowia pulcherrima, Kluyveromyces lactis, and Candida sake. An integrative parameter, Efficacy (efficacy for alcohol level reduction) was designed to simplify comparisons between strains or growth conditions. It integrates sugar consumption, ethanol yield, and acetic acid production data. We found a high relative impact of nitrogen source availability and temperature, as compared to aeration conditions, for several fermentation parameters, including ethanol yield. However, increasing oxygen supply showed a positive impact in terms of alcohol reduction and Efficacy for all the strains tested. The best results across assays were obtained for C. sake CBS 5093, with high sugar consumption rates, associated to low ethanol yields, and very low acetic acid production. Processes involving this yeast strain would benefit from high aeration levels and low nitrogen source availability; while fermentation temperatures would have little impact on its Efficacy for alcohol level reduction.
    Full-text · Article · Jan 2016
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    • "All these results are in contrast to those by Aceituno et al. (2012) who described acetic acid production to take place only under fully anaerobic conditions. Probably, the use of nitrogen-limited chemostat by Aceituno et al. (2012) is related to the differences they found in the pattern of acetic acid production, as compared to other authors. "
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    ABSTRACT: We have developed a wine fermentation procedure that takes advantage of the metabolic features of a previously characterized Metschnikowia pulcherrima strain in order to reduce ethanol production. It involves the use of M. pulcherrima/Saccharomyces cerevisiae mixed cultures, controlled oxygenation conditions during the first 48 h of fermentation, and anaerobic conditions thereafter. The influence of different oxygenation regimes and initial inoculum composition on yeast physiology and final ethanol content was studied. The impact of oxygenation on yeast physiology goes beyond the first aerated step and influences yields and survival rates during the anaerobic stage. The activity of M. pulcherrima in mixed oxygenated cultures resulted in a clear reduction in ethanol yield, as compared to S. cerevisiae. Despite relatively low initial cell numbers, S. cerevisiae always predominated in mixed cultures by the end of the fermentation process. Strain replacement was faster under low oxygenation levels. M. pulcherrima confers an additional advantage in terms of dissolved oxygen, which drops to zero after a few hours of culture, even under highly aerated conditions, and this holds true for mixed cultures. Alcohol reduction values about 3.7 % (v/v) were obtained for mixed cultures under high aeration, but they were associated to unacceptable volatile acidity levels. In contrast, under optimized conditions, only 0.35 g/L acetic acid was produced, for an alcohol reduction of 2.2 % (v/v), and almost null dissolved oxygen during the process. Electronic supplementary material The online version of this article (doi:10.1007/s00253-014-6321-3) contains supplementary material, which is available to authorized users.
    Full-text · Article · Jan 2015 · Applied Microbiology and Biotechnology
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    • "Gene expression Two additional parameters were included to account for transcriptomic (gene expression) information. Eighteen normalized microarray expression experiments (6 for aerobic and 12 for anaerobic cultivations) of different yeast strains growing on glucose were obtained (a) from a published work (Lai et al., 2006) using the query-driven search tool SPELL (Serial Pattern of Expression Levels Locator) (Hibbs et al., 2007) from the Saccharomyces Genome Database (SGD) (Cherry et al., 2012), and (b) from two works from our group (Aceituno et al., 2012; Orellana et al., 2014). All data was preprocessed, disregarding genes that were not included in Yeast 5 or that were essential for biomass growth, using single-knockout gene analysis (Edwards and Palsson, 2000; Zomorrodi et al., 2012). "
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    ABSTRACT: Dynamic flux balance analysis (dFBA) has been widely employed in metabolic engineering to predict the effect of genetic modifications and environmental conditions in the cell's metabolism during dynamic cultures. However, the importance of the model parameters used in these methodologies has not been properly addressed. Here, we present a novel and simple procedure to identify dFBA parameters that are relevant for model calibration. The procedure uses metaheuristic optimization and pre/post regression diagnostics, fixing iteratively the model parameters that do not have a significant role. We evaluated this protocol in a Saccharomyces cerevisiae dFBA framework calibrated for aerobic fed-batch and anaerobic batch cultivations. The model structures achieved have only significant, sensible and uncorrelated parameters and are able to calibrate different experimental data. We show that consumption, suboptimal growth and production rates are more useful for calibrating dynamic S. cerevisiae metabolic models than Boolean gene expression rules, biomass requirements and ATP maintenance.
    Full-text · Article · Sep 2014 · Metabolic Engineering
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