*2.3. Fermentation Experiments*

The fermentations assays were carried out according to the different experimental lines described by the DoE (Table 3). Therefore, sugar beet molasses was diluted to reach the required initial sugar concentration using tap water and the respective amount of nutrient (Fermaid O, Lallemand Biofuels & Distilled Spirits, Montreal, QC, Canada) was added to the solution. Fermaid O is a mix of inactivated yeast fractions, rich in organic nitrogen, generally used in the wine industry. This nutrient was not supplemented by any ammonia salts or micronutrients. Fermaid O was referred to as "nutrient" parameter in this study. After pH adjustment to 5.5 using an 11 M H2SO<sup>4</sup> solution, fermentation broths were placed into 50 mL serum vials with a working volume of 25 mL. Yeast inoculum was prepared by rehydrating *Saccharomyces cerevisiae* dry yeast cells (Thermosacc® DRY, Lallemand Biofuels & Distilled Spirits, Canada) with tap water at 30 ◦C, 140 rpm for a period of 15 min using a shaking incubator (Ecotron, Infors-HT Inc., Bottmingen, Switzerland). Three concentrated yeast suspensions were prepared to obtain the desired initial yeast concentration in the final fermentation media. After yeast inoculation, all the fermentation vials were capped with rubber septum stoppers and aluminium rings, before being flushed with N<sup>2</sup> for 5 min to ensure an anaerobic environment. The fermentation vials were finally incubated at 30 ◦C for 112 h under a 140 rpm stirring. Temperature and agitation parameters were not optimised in this work and their values were selected based on our previous study dealing with the fermentation of softwood residues [21]. Liquid samples were taken at the beginning and at the end of the experiment to quantify different metabolites and products such as sugars and ethanol concentrations. All conditions were carried out in triplicate and results are expressed as average value ± standard deviation.


**Table 3.** Experimental design matrix of the CCD-RSM study presenting the different experimental lines as well as the investigated response generated during the molasses fermentation optimization.

> The optimal conditions obtained during the CCD were used to design and perform scale-up assays using 7.5 and 100 L bioreactors with working volumes of 5 and 50 L, respectively (Figure 1). The 7.5 L glass bioreactor (Infors-HT Inc., Bottmingen, Switzerland) was equipped with a double envelope to control the temperature at 30 ◦C, meanwhile, a mechanical stirrer provided a continuous agitation at 250 rpm. In addition, a watercooled condenser was attached to the reactor to prevent evaporation. The 100 L stainless bioreactor was equipped with a 2000 W heating element to maintain the fermentation media temperature to 30 ◦C. Constant stirring at 70 rpm was achieved using a 2-blades impeller system powered by a 12 Volt DC electric motor.

**Figure 1.** Schematic view of the 7.5 L (**A**) and 100 L (**B**) stirred-tank bioreactors used to perform the fermentation scale-up experiments. The optimal operating conditions determined during the CCD-RSM were utilised to maximise the production of ethanol from non-treated sugar beet molasses.
