*3.4. RSM Optimization*

The RSM analysis highlighted the combined effect of the three fermentation process parameters on the ethanol productivity response (Figure 4). As observed previously, nutrient supplementation was one of the most influential variables, strongly affecting the fermentation performances. Optimal regions for maximizing the overall ethanol productivity (>15 g/L/d) were detected for a nutrient concentration of 4 g/L combined with an initial sugar concentration lower than 225 g/L and a minimum of 0.2 g/L of yeast (Figure 4C). In the absence of nutrient, ethanol production rates were very slow (<10.6 g/L/d), suggesting that the molasses feedstock alone did not offer a sufficient level of nutrient for *S. cerevisiae* yeast metabolism. These findings are slightly different from other studies reporting the efficient production of ethanol from sugar beet molasses without nutrient supplementation [13,23]. However, yeast cells used in these studies were previously grown in a nutrient-rich solution before inoculation, which was very different from our simple rehydration yeast preparation protocol.

In addition, ethanol productivity performances were negatively affected when increasing the amount of initial sugar present in the fermentation system, which was probably due to an increase in osmotic stress. This behaviour was similarly reported by Vuˇcurovi´c et al. when measuring *S. cerevisiae* yeast viability at the end of sugar beet molasses fermentation assays. In the absence of nutrient, the increase of initial sugar concentration from 100 to 300 g/L significantly decreased the viability of free cells from around 100 to 63.9% [17].

**Figure 4.** 3D-response curves of the ethanol productivity as a function of yeast and sugar concentrations for the three investigated nutrient concentrations of 0 (**A**), 2 (**B**) and 4 g/L (**C**). Untreated sugar beet molasses, diluted at several initial sugar concentration, was used as the fermentation feedstock. Red dots within the response curves represent experimental design points obtained during the CCD-RSM while contour lines show prediction outputs calculated by the mathematical model.
