2.4.1. Gravimetric Analysis

To monitor the ethanol production of a large number of fermentation media during the CCD-RSM, a gravimetric methodology was developed to quantify the amount of CO<sup>2</sup> released during the fermentation procedure. Considering the alcoholic fermentation reaction (Equation (2)) that theoretically gives two moles of ethanol and two moles of CO<sup>2</sup> per initial mole of glucose, the ethanol concentration could be indirectly calculated by following the production of CO2. This gravimetric technique is a simple and efficient tool to follow ethanol production kinetics, with the advantage to be less time- and resourceconsuming than conventional methods. Moreover, this technique assumes that only CO<sup>2</sup> and ethanol are produced during fermentation.

To validate this technique, a calibration curve was prepared by plotting "the ethanol calculated by mass loss" versus "the ethanol quantified by HPLC" (details in Section 2.4.2), which were referred to as Eml and Ehplc, respectively (Figure S1). The linear regression showed a coefficient of determination (R<sup>2</sup> ) of 0.97 with Equation (3).

$$\rm{E\_{hplc}} = 0.84 \times \rm{E\_{ml}} - 9.95 \tag{3}$$

All the ethanol results shown in this study, except for the ones obtained during the scale-up experiments, were calculated using the latest calibration curve.
