Surveyor plus chromatograph

Production and consumption of the main fermentation-related metabolites in daily samples, (glucose, fructose, glycerol, acetic acid and ethanol) were determined in duplicate using a Surveyor Plus chromatograph (Thermo Fisher Scientific, Waltham, MA) equipped with a refractive index and a photodiode array detector (Surveyor RI Plus and Surveyor PDA Plus, respectively). Hyper REZ XP carbohydrate H+8 μm column and guard (Thermo Fisher Scientific) were used and maintained at 50°C. Elution was performed with 1.5 mM H 2 SO 4 as mobile phase, at a flow rate of 0.6 mL/min.
Prior to injection, samples were filtered through 0.22-μm-pore-size nylon filters and diluted 10-fold.
One way analysis of variance was carried out on the main fermentation metabolites. Means of biological replicates were compared using Tukey's test, with significance level set at 5%. All analyses were performed using SPSS Statistics v. 23 program (IBM, Armonk, NY).

Extracellular glucose, fructose, glycerol and ethanol were analyzed in all the supernatant samples during steady states. Analytical HPLC was carried out in a Surveyor Plus Chromatograph (Thermo Fisher Scientific, Waltham, MA) equipped with a refraction index detector, an autosampler and a UV-Visible detector. Prior to injection, samples were centrifuged at 13300 rpm for 5 min, and samples were diluted 10-fold and filtered through 0.22 µm pore size nylon filters (Micron Analitica, Spain). A total volume of 25 L was injected into a HyperREZ XP Carbohydrate H+8 mm column (Thermo Fisher Scientific) assembled to its correspondent guard. The mobile phase used was 1.5 mM H 2 SO 4 with a flux of 0.6 mL min -1 and a column temperature of 50°C. The concentration of each compound was calculated using external standards.
Each sample was analyzed in duplicate.