*3.2. Design Matrix and Ethanol Productivity Analysis*

The fermentation results obtained during the CCD-RSM were analysed by calculating the ethanol productivity of each experimental line, based on the maximum ethanol concentration achieved after a specific incubation time (Table 3). To quantify the fermentation performances and perform an in-depth comparison of the results, ethanol yields expressed as a percentage of the maximum theoretical ethanol yield were calculated and discussed in the following section.

As observed for the fermentation kinetics, various responses were detected among the experimental lines of the CCD-RSM, with ethanol productivity values ranging from 2.6 to 19.8 g/L/d with average productivity of 13.2 g/L. A total of 8 experiments achieved ethanol productivities higher than 15.0 g/L by the end of incubation time.

However, ethanol yields calculated for all the 16 experimental runs revealed that only a few numbers of conditions reached high ethanol yields (Figure 3). In fact, the initial sugar concentration had an important effect on the fermentation performances since only the experiments with sugar concentrations comprised between 125 and 225 g/L reached ethanol yields above 60%. In addition, the highest ethanol yields of 82% were generated for three experimental conditions (run 8, 5 and 2), where a minimum of 2 g/L of nutrient and 0.2 g/L of yeast were used, combined with an initial sugar concentration of 125 g/L. Finally, the lower ethanol yield of 7.0% was obtained for the fermentation media incubated without any nutrient supplementation combined with a low amount of yeast (0.2 g/L) and a high concentration of initial sugar (325 g/L).

**Figure 3.** Ethanol yields expressed as a percentage of the maximum theoretical ethanol concentration for all the investigated runs of the CCD-RSM (white label). Yields were sorted in ascending order for a better interpretation of the ethanol performances. Dashed lines show the lowest (Min), the highest (Max) and the average (Mean) ethanol yields.

The high ethanol volumetric productivity values obtained in our study, ranging from 15.0 to 19.8 g/L/d, were in agreement with the fermentation results previously presented by Razmovski et al. using sugar beet molasses [13]. The use of non-immobilised yeast cells generated ethanol volumetric productivities of 20.09, 19.82 and 13.51 g/L/d when using initial sugar concentration of 100, 150 and 300 g/L, respectively. Similarly, Vucurovic et al. noticed a significant reduction in ethanol performances including sugar utilization, ethanol productivity and yield when fermenting sugar beet molasses with sugar concentration higher than 175 g/L, using free yeast cells [23]. These observations were correlated with lower cell yeast viability, attributed to the presence of high sugar concentration as well as non-sugar compounds such as mineral salts which might affect yeast metabolism.

The analysis of experimental ethanol yields was useful to correlate the ethanol productivity values with actual ethanol performances based on the maximum theoretical ethanol yield. The latest parameter is often calculated considering 100% sugar utilization by yeast cells during fermentation. Such operational parameter must be evaluated when implementing a fermentation process in order to take full advantage of the initial sugars present in the feedstock for the production of ethanol. In this sense, sugar concentration between 125 and 225 g/L and the addition of nutrient should be carefully considered to achieve high ethanol yields while producing at least 15 g/L/d of ethanol.
