## 3 Results and Discussion
### Comparative Analysis of SHF and SSF Configurations for 2,3-BDO Production from Carrot Discard
[3] found that while _Paenibacillus polymyxa_ DSM 365 could co-utilize glucose and fructose to some extent, galactose consumption was significantly delayed until glucose was nearly exhausted, being the glucose utilization 10.7 times greater than that of galactose. In other work, diauxic growth patterns have also been clearly demonstrated during the co-fermentation of glucose and pentoses [5]. The results of our study thus align perfectly with these established metabolic behaviors. This resulted in a final 2,3-BDO production of 10.9 g/L, a yield of 0.17 g/g, and a productivity of 0.236 g/L-h (Table 3, Figure 3b). Notably, this strain co-produced a substantial acetoin titer of 24.2 g/L (Table 3, Figure 3c), alongside lower concentrations of ethanol (0.4 g/L) (Table 3, Figure 3d), acetic acid (2.2 g/L) (Figure S5b), and lactic acid (2.5 g/L) (Figure S5a). In stark contrast, the SSF strategy was ineffective. Sugar metabolism was delayed by 6-8 h and ultimately stalled, resulting in only 51% sugar uptake after 144 h (Figure 3a). Consequently, 2,3-BDO production was minimal (0.9 g/L). This poor performance is likely attributable to cellular stress from the high solid load and low water content, as well as osmotic pressure in the SSF environment, which is known to inhibit bacterial growth and metabolic function [27]. In this way, according to [PERSON] et al. [28], the high solid content in the process could result in deteriorated enzyme and microorganism activities and an increase in viscosity, hindering the homogeneous and effective distribution of the enzymes and microorganisms. Moreover, in the SSF configuration, the fermentation began with a heterogeneous slurry where sugars were not immediately bioavailable. Then, this fact could also interfere negatively with the metabolism of both _Bacillus_ strains. The metabolic response to these challenging conditions was evident in our results. For both _B. licheniformis_ and _B. amyloliquefaciens_, the metabolic flux during SSF shifted decisively away from 2,3-BDO production and towards the synthesis of organic acids, resulting in high titers of lactic acid (11.1-12.5 g/L) and acetic acid (5.1-5.3 g/L) (Figure S5). This shift towards organic acid production under stressful SSF conditions is consistent with findings in the literature, where SSF processes have been effectively employed to produce lactic acid from corn crop residues by _Bacillus coagulus_[29] and succinic acid from corn stover using _Actinobacillus succinogenes_[30]. Therefore, the SHF configuration was unequivocally superior for _B. licheniformis_. Similarly, when using _B. amyloliquefaciens_ in the SHF configuration, all sugars were consumed by the end of fermentation (t = 144 h), with over 80% uptake achieved by 56 h (Figure 3a). This strain also preferentially consumed glucose (72 h) over other sugars (144 h) (Figure S4). The SHF process yielded a high 2,3-BDO production of 16.7 g/L within 56 h, corresponding to a yield of 0.33 g/g and a productivity of 0.298 g/L-h (Table 3, Figure 3b). Co-produced acetoin reached 8.0 g/L (Table 3, Figure 3c), while ethanol was minimal (0.2 g/L) (Table 3, Figure 3d) and organic acids were low (3.1-3.4 g/L) (Figure S5). As observed with the other strain, the SSF configuration was markedly less efficient for _B. amyloliquefaciens_. Only 46.4% of the available sugar was consumed, yielding just 4.1 g/L of 2,3-BDO and 3.1 g/L of acetoin (Table 3 and Figure 3). Again, metabolism shiftedtowards organic acids (12.5 g/L lactic acid and 5.3 g/L acetic acid) (Figure S5), indicating process inhibition [27]. Thus, SHF was also the best strategy for this strain. The behavior of pH was also very different for SHF and SSF configurations (Figure S1c), in both fermentations by _B. licheniformis_ and _B. amyloliquefaciens_. While pH decreased (up to 4.2-4.4) during the whole fermentation in SSF, probably due to the formation of lactic and acetic acids, in SHF, a decrease was observed during the first hours of the process (up to 5.6-5.7 at 22 h), increasing after this fermentation time.