The extracellular glycerol concentrations and residual sugars (glucose and fructose) were determined in must and medium samples by HPLC (Thermo Fisher Scientific, Waltham, MA) equipped with a refraction index detector. The column employed was a HyperREZTM XP Carbohydrate H+ 8 μm (Thermo Fisher Scientific) and the conditions used in the analysis were as follows: eluent, 1.5 mM H2SO4; flux, 0.6 ml/min; and oven temperature, 50°C. The samples were diluted, filtered through a 0.22-μm nylon filter (Symta, Madrid, Spain) and injected in duplicate.
To determine intracellular glycerol content, 5 OD600 units were harvested by filtration and quickly washed with 5 ml of water and transferred to a tube containing 1 ml of cold water. After no more than 20 s after sampling, the yeast suspension was boiled for 10 min, cooled on ice, and centrifuged at 15,300 × g for 10 min (4°C). The supernatant was collected, filtered and directly analyzed by HPLC. A second sample (5 OD600 units) was harvested by filtration in cellulose membrane, 25 mm pore size 0.45 μm (MF-Milipore) previously dried in the microwave at 350W for 20 min. and weighed. To determine dry weight, the cells in the membrane were carefully washed with 1 ml of water and dried under the same conditions. The values obtained are expressed as μg of glycerol per mg of yeast cells. Experiments were performed in triplicate.
To determine intracellular glycerol content, 5 OD600 units were harvested by filtration and quickly washed with 5 ml of water and transferred to a tube containing 1 ml of cold water. After no more than 20 s after sampling, the yeast suspension was boiled for 10 min, cooled on ice, and centrifuged at 15,300 × g for 10 min (4°C). The supernatant was collected, filtered and directly analyzed by HPLC. A second sample (5 OD600 units) was harvested by filtration in cellulose membrane, 25 mm pore size 0.45 μm (MF-Milipore) previously dried in the microwave at 350W for 20 min. and weighed. To determine dry weight, the cells in the membrane were carefully washed with 1 ml of water and dried under the same conditions. The values obtained are expressed as μg of glycerol per mg of yeast cells. Experiments were performed in triplicate.