2.4.2. Sugars and Ethanol Quantification

Although the gravimetric analysis is an alternative option to follow the fermentation development, it is still necessary the quantify monomeric sugars and other fermentationrelated metabolites to ensure accurate results. To this purpose, a Dionex ICS-5000+ ion chromatography system was used but more specifically for sugars quantification. The system was equipped with a KOH eluent generator to provide a proper eluent concentration,

an analytical gradient pump, a thermostatic AS-AP auto-sampler and an electrochemical detector. A 200 mM KOH post-injection with a Dionex GP 50 gradient pump was implemented to ensure signal stability. For this project, a Dionex CarboPac Sa10-4 µM column was used while the oven was set at 45 ◦C and the electrochemical detector was at 30 ◦C. The injection volume was 0.4 µL and elution was made with an aqueous solution of KOH at 1.25 mL/min at the following concentrations: 1 mM for 12 min, 10 mM for 5 min and 1 mM for 1 min. The calibration curve involved a concentration of standards varying from 10 ppm to 1000 ppm and involved the following standards: fructose 99%, glucose 99% and sucrose 99% which were all purchased from Sigma-Aldrich.

An Agilent 1100 series HPLC (Agilent Technologies Inc., Colorado Springs, CO, USA) equipped with a G1362A Refractive Index Detector was used to quantify ethanol. The system was operated at 40 ◦C with an isocratic elution method (2.5 mM). The HPLC set-up also had a G1322A Degasser and a G1311A Quaternary Pump. A G1313A Autosampler injected 40 µL of sample and the column used was a ROA-Organic Acid H+ (8%) at 65 ◦C. The elution was performed at a constant flow of 0.08 mL/min of a 0.01 M H2SO<sup>4</sup> solution. A calibration curve from 10 ppm to 1000 ppm was performed using the following standards: L-lactic 99% (Alfa Aesar), acetic acid 99.9% (Sigma Aldrich), ethanol 99% (Sigma Aldrich) and glycerol 99% (Sigma Aldrich).
