Effect of Myclobutanil Pesticide on the Physiological Behavior of Two Newly Isolated Saccharomyces cerevisiae Strains during Very-High-Gravity Alcoholic Fermentation

Abstract

Yeasts are able to act as biosorbents, as their cell wall includes several components capable of binding organic xenobiotic compounds that can potentially be removed during various fermentation processes. In the present investigation, two novel Saccharomyces cerevisiae strains (LMBF-Y 16 and LMBF-Y-18), previously isolated from grapes, were studied regarding their physiological behavior (dry cell weight-DCW production, substrate uptake, and ethanol and glycerol biosynthesis) during fermentations of grape must, in some cases enriched with commercial glucose and fructose (initial total sugar concentration approximately 150 and 250 g/L, respectively). Myclobutanil (a chiral triazole fungicide broadly used as a protective agent of vine) was also added to the culture media at various concentrations in order to assess the ability of the yeasts to simultaneously perform alcoholic fermentations and detoxify the medium (i.e., to remove the fungicide). In the first set of experiments and for both tested strains, trials were carried out in either 250 mL or 2.0 L agitated shake flasks in either synthetic glucose-based experiments or grape musts. Since the results obtained in the trials where the cultures were placed in 2.0 L flasks with grape musts as substrates were superior in terms of both DCW and ethanol production, these experimental conditions were selected for the subsequent studies. Both strains showed high fermentative efficiency, producing high amounts of DCW (9.5-10.5 g/L) in parallel with high ethanol production, which in some cases achieved values very close to the maximum theoretical ethanol production yield (≈0.49 g of ethanol per g of sugar). When using grape must with initial total sugars at approximately 250 g/L (very high gravity fermentation media, close to winemaking conditions), significantly high ethanol quantities (i.e., ranging between 105 and 123 g/L) were produced. Myclobutanil addition slightly negatively affected sugar conversion into ethanol; however, in all cases, ethanol production was very satisfactory. A non-negligible myclobutanil removal during fermentation, which ranged between 5%-27%, as a result of the adsorptive or degradative capacity of the yeast was also reported. The presence of myclobutanil had no effect on DCW production and resulted in no significant differences in the biosynthesis of glycerol. Therefore, these newly isolated yeast strains could be excellent candidates for simultaneous high ethanol production and parallel pesticide removal in a general biorefinery concept demonstrating many environmental benefits.

Keywords: Saccharomyces cerevisiae; ethanol production; myclobutanil; very-high-gravity fermentation.

Figure 1

Electrophoretical patterns obtained for the two tested yeast strains, LMBF-Y16 and LMBF-Y18, with delta12–delta21 primers. The commercial Saccharomyces cerevisiae strain VL3 (Laffort) was used as a positive control. A 100 bp DNA ladder marker (BioRad) served as the size standard.

Figure 2

Kinetics of biomass (yeast dry cell weight, DCW) (X, g/L) (a), ethanol (Eth, g/L) (b), and total sugars (TS, g/L) (c) during growth of Saccharomyces cerevisiae strain LMBF-Y 16 on sugar-enriched grape must with initial total sugar concentration (TS₀) adjusted to c. 220 g/L. Culture conditions: growth on 2.0 L flasks previously pasteurized (10 min, T = 95 °C), at 180 ± 5 rpm, pH value throughout the culture = 3.5 ± 0.2, incubation temperature T = 25 ± 1 °C. Three replications of the same experiment are presented. Each experimental point presented in the runs is the mean value of two independent measurements (SE < 15%).

Figure 3

Evolution of total sugars (TS, g/L) (a) and ethanol (Eth, g/L) (b) during growth of Saccharomyces cerevisiae strain LMBF-Y 16 on either glucose-based salt-enriched synthetic media or sugar-enriched grape must with initial total sugar concentration (TS₀) ≈ 220 g/L. Culture conditions: growth on 250 mL flasks previously pasteurized (10 min, T = 95 °C) at 180 ± 5 rpm, pH value throughout the culture = 3.5 ± 0.2, incubation temperature T = 25 ± 1 °C. Each point is the mean value of two independent measurements (SE < 15%). Each point is the mean value of two independent measurements (SE < 15%).

Figure 3

Evolution of total sugars (TS, g/L) (a) and ethanol (Eth, g/L) (b) during growth of Saccharomyces cerevisiae strain LMBF-Y 16 on either glucose-based salt-enriched synthetic media or sugar-enriched grape must with initial total sugar concentration (TS₀) ≈ 220 g/L. Culture conditions: growth on 250 mL flasks previously pasteurized (10 min, T = 95 °C) at 180 ± 5 rpm, pH value throughout the culture = 3.5 ± 0.2, incubation temperature T = 25 ± 1 °C. Each point is the mean value of two independent measurements (SE < 15%). Each point is the mean value of two independent measurements (SE < 15%).

Figure 4

Evolution of dissolved oxygen tension (DOT, % v/v) during growth of Saccharomyces cerevisiae strain LMBF-Y 16 on either glucose-based, salt-enriched synthetic media or sugar-enriched grape must with initial total sugar concentration ≈ 220 g/L. Each point is the mean value of two independent measurements (SE < 15%).

Figure 5

Biomass (yeast dry cell weight) (X, g/L) evolution during growth of Saccharomyces cerevisiae strain LMBF-Y 18 on grape-must-based media with initial total sugar concentration (a) ~150 g/L and (b) ~250 g/L, with or without the addition of myclobutanil fungicide at different concentrations (0.1 mg/L and 1.0 mg/L). Culture conditions as in Table 3. Each point is the mean value of two independent measurements (SE < 15%).

Figure 6

Total sugars (TS, g/L) evolution during growth of Saccharomyces cerevisiae strain LMBF-Y 18 on grape-must-based media with initial total sugar concentration (a) ~150 g/L and (b) ~250 g/L, with or without the addition of myclobutanil fungicide at different concentrations (0.1 mg/L and 1.0 mg/L). Culture conditions as in Table 3. Each point is the mean value of two independent measurements (SE < 15%).

Figure 7

Ethanol (Eth, g/L) evolution during growth of Saccharomyces cerevisiae strain LMBF-Y 18 on grape-must-based media with initial total sugar concentration (a) ~150 g/L and (b) ~250 g/L, with or without the addition of myclobutanil fungicide at different concentrations (0.1 mg/L and 1.0 mg/L). Culture conditions as in Table 3. Each point is the mean value of two independent measurements (SE < 15%).

Figure 7

Ethanol (Eth, g/L) evolution during growth of Saccharomyces cerevisiae strain LMBF-Y 18 on grape-must-based media with initial total sugar concentration (a) ~150 g/L and (b) ~250 g/L, with or without the addition of myclobutanil fungicide at different concentrations (0.1 mg/L and 1.0 mg/L). Culture conditions as in Table 3. Each point is the mean value of two independent measurements (SE < 15%).