*3.6. Scale-Up Assays*

Scaling up biological processes such as the alcoholic fermentation process often requires further investigation at an intermediate scale to realise a smooth transition from bench-scale to pilot-scale. In this context, experimental results obtained in the first part of this study were used to design and perform fermentation assays at bioreactor-scale. Diluted sugar beet molasses, with an initial total sugar concentration of 150 g/L, were used as feedstock to perform batch fermentation using two bioreactors with total volumes of 7.5 and 100 L, respectively. Fermentation broths were regularly sampled to monitor the sugar and ethanol concentration throughout the experiment.

During the first 24 h of incubation, the diminution of sucrose concentration was correlated with the production of 22.5 g/L of ethanol as well as an increase of glucose and fructose concentrations, for both the 7.5 and 100 L bioreactors (Figure 6). The latter phenomena can certainly be attributed to the hydrolysis of sucrose by *S. cerevisiae*'s invertase enzyme [24]. It is worth mentioning that glucose was preferentially utilised by yeast cells, with a higher consumption rate than fructose. Then, the concentration of both reducing sugars continuously decreased over the fermentation and after 64 h of reaction, could not be detected anymore.

**Figure 6.** Ethanol and sugar concentration variations during fermentation scale-up assays performed in 7.5 L (**A**) and 100 L (**B**) batch bioreactors. Non-treated diluted sugar beet molasses with a total initial sugar concentration of 145 g/L was used for this experiment with 4.0 g/L of nutrient and 0.27 g/L of yeast. The dashed line refers to the theoretical maximum ethanol yield.

The two bioreactors presented slightly different behaviours in term of ethanol productivity as well as sugar utilization rate. For the 7.5 L bioreactor, ethanol concentration increased from 65.6 to 79.6 g/L between 48 and 64 h of fermentation, corresponding to a variation of ethanol yields from 81.9% and 99.5%, respectively. Interestingly, the ethanol concentration measured in the 100 L bioreactor remained similar between 48 and 64 h of incubation (around 65 g/L of ethanol), even if the fructose concentration decreased from 18.4 to 0 g/L, after 16 h. The latest observation suggests that some ethanol might have been produced during the last hours of incubation but directly escaped the fermentation broth due to evaporation. This situation could be explained by the fact that the 100 L bioreactor was not equipped with a water-cooled condenser system as compared to the 7.5 L bioreactor.

In terms of ethanol productivity, the 7.5 L bioreactor generated 29.9 g/L/d of ethanol compared with 23.7 g/L/d for the 100 L bioreactor when calculating the productivity after 64 h of incubation (Table 5). The lower fermentation performances observed for the 100 L bioreactor highlights the risk of ethanol evaporation during the scale-up of fermentation processes. In addition, analysis of the final fermentation broths showed very low levels of organic compounds such acetic and lactic acids, which confirmed that the scale-up experiments were not subjected to any major microbial contamination.

**Table 5.** Fermentation performances and by-products concentrations measured after 64 h of incubation at 7.5 and 100 L bioreactors scale using diluted sugar beet molasses (170 g/L of initial sugar) together with 0.27 g/L of *Saccharomyces cerevisiae* and 4.0 g/L of nutrient.


The ethanol productivity values obtained during the scale-up assays were found to be significantly higher than the ones obtained at lab-scale using the 50 mL vials, probably due to the difference of scale and reactor configuration. The most important difference between the incubator and bioreactor scales might be related to the mixing conditions, impacting the mass transfer between the yeast cells and the fermentation broth. The agitation performed in the incubator with the 50 mL fermentation vials was indirectly using an orbital shaker, which was certainly less efficient than the direct agitation maintained by a mechanical impeller, in the case of the 7.5 and 100 L reactors. In the case of the bioreactors, the microorganisms were continuously in suspension which could have significantly increased their contact with the molasses, resulting in a higher sugar utilization rate and ethanol productivity. In a recent study, the production of ethanol using raw cassava starch was investigated from a 5 L laboratory and a 200 L pilot scale up to a 3000 L industrial-scale system [25]. Although the technology and dimension of the three latter bioreactors were different, very comparable ethanol productivities and kinetics parameters were obtained for the three scales by adjusting the power input, also known as the energy dissipation rate per unit mass, to a specific value of 0.10 W/kg. Consequently, the agitation rates of the 5, 200 and 3000 L bioreactors were respectively fixed at 200, 125 and 55 rpm based on the fermenter dimension, impeller type and size.

In addition to the mixing differences, the fermentation vials used during the CCD-RSM were sealed and flushed with nitrogen while the bioreactors were let at atmospheric pressure without nitrogen sparging. Thus, the build-up of pressure accumulated within the small fermentation vials could have affected the yeast metabolism, lowering the ethanol production performances. Similar findings were reported when investigating the effect of different process parameters on the fermentation of apple pomace by co-culturing filamentous fungi with yeast [26]. At an inoculation rate of 4.0% (*v*/*v*) of *S. cerevisiae* combined with 4.0% (*w*/*v*) of *T. harzianum* and *A. sojae*, increasing the agitation speed from 0 to 200 rpm showed a significant increase in the ethanol production when employing non-sealed reactor vessels.

These results highlighted the challenges associated with the scale-up of fermentation processes from lab to bioreactor scales, especially regarding the impact of agitation speed and reactor dimension. In another study, the scale-up of bioethanol production from soybean molasses raised different problems, especially with the formation of foam and the risk of bacterial contamination at pilot and industrial scales [27].
