*3.1. Ethanol Production Kinetics Overview*

One of the main objectives of this study was to elucidate the effect of different operating fermentation parameters on the ethanol production process to develop a cost-effective and efficient strategy to valorise sugar beet molasses. In this sense, a CCD-RSM was designed and developed using three experimental process parameters (initial sugar, yeast and nutrient concentrations) investigated at three levels, using ethanol productivity as the main process response.

Ethanol production was strongly affected by the three investigated process parameters, which resulted in different fermentation kinetics among the 16 experiments of the CCD-RSM (Figure 2). For all the conditions, a short lag phase of approximately 24 h was noticed before measuring any production of ethanol. This period is probably due to the adaptation of yeast cells after their inoculation into the sugar-rich media. By the end of the incubation time, only three conditions with an initial sugar concentration of 125 g/L, various yeast (0.2, 0.6 and 1.0 g/L) and nutrient (2 and 4 g/L) concentrations reached a plateau with a constant ethanol concentration of 54 g/L. The highest ethanol production of 92 g/L was observed when the sugar beet molasses was diluted to 225 g/L of initial sugars combined with the use of 0.6 g/L and 4.0 g/L of yeast and nutrient, respectively. The positive effect of nutrient supplementation was highlighted by observing very slow fermentation kinetics for media not supplemented with nutrients compared with the ones with nutrients.

**Figure 2.** Ethanol production for the different initial sugar concentration of sugar beet molasses (125, 225 and 325 g/L) using *Saccharomyces cerevisiae* yeast. Colours are attributed to the initial nutrient concentration: black (0.0 g/L), blue (2.0 g/L) and red (4.0 g/L). Symbols are related to the initial yeast concentration: square (0.2 g/L), circle (0.6 g/L) and triangle (1.0 g/L).
