## 3 Results and Discussion
### 2,3-BDO Production from Glucose Semi-Defined Media
#### 3.1.2 2,3-BDO Fermentation by _B. amyloliquefaciens_  
To provide a comparative analysis, fermentation assays were conducted with _B. amyloliquefaciens_ under the same conditions used for _B. licheniformis_, with initial glucose concentrations ranging from 20 to 120 g/L. The results are presented in Table 2 and Figure 2.  
_B. amyloliquefaciens_ achieved high glucose consumption (\(\geq\)90%) by the end of fermentation (t = 144 h) in cultures with initial concentrations up to 80 g/L (Table 2). However, a further increase in the initial glucose concentration resulted in substantial residual substrate, with up to 60% remaining unconsumed in the 120 g/L culture (Figure 2a). Correspondingly, the time required for maximum substrate utilization increased with the initial glucose load, reaching up to 56 h for the G20-G80 cultures. Maximum 2,3-BDO concentrations rose from 5.8 to 19.8 g/L as the initial glucose concentration was increased from 20 to 100 g/L (Table 2, Figure 2b). However, increasing the initial concentration to 120 g/L resulted in a 24.2% decrease in the final 2,3-BDO production. This suggests a strong substrate inhibition effect on _B. amyloliquefaciens_ at glucose concentrations exceeding 100 g/L. Notably, after glucose depletion, the produced 2,3-BDO was subsequently re-consumed by the microorganism in all tested conditions (Figure 2b). \begin{table}
\begin{tabular}{c c c c c c c c} \hline \hline
**Initial Glucose Conc.** & **Time** & **Sugar Uptake** & **2,3-BDO** & **Acetoin** & **Biomass OD** & **Y\({}_{\text{2,3-BDO/sugars}}\)** & **P\({}_{\text{2,3-BDO}}\)** \\ **(g/L)** & **(h)** & **(\%)** & **(\%)** & **(g/L)** & **(g/L)** & **(600 nm)** & **(g/g)** & **(g/L h)** \\ \hline
20 & 22 & 100 (100) & 5.8 \(\pm\) 0.0 & 3.3 \(\pm\) 0.3 & 9.8 \(\pm\) 0.6 & 0.29 & 0.265 \\
40 & 32 & 79.7 (100) & 11.1 \(\pm\) 0.3 & 3.5 \(\pm\) 0.3 & 13.9 \(\pm\) 0.3 & 0.34 & 0.348 \\
60 & 56 & 92.8 (100) & 16.9 \(\pm\) 0.0 & 9.1 \(\pm\) 0.0 & 14.4 \(\pm\) 0.1 & 0.31 & 0.302 \\
80 & 56 & 62.3 (90.5) & 15.5 \(\pm\) 0.0 & 7.6 \(\pm\) 0.1 & 12.9 \(\pm\) 0.8 & 0.32 & 0.276 \\
100 & 72 & 61.0 (74.2) & 19.8 \(\pm\) 0.0 & 10.3 \(\pm\) 0.0 & 8.6 \(\pm\) 0.3 & 0.32 & 0.275 \\
120 & 56 & 32.2 (39.7) & 15.0 \(\pm\) 1.0 & 6.8 \(\pm\) 0.4 & 8.0 \(\pm\) 0.5 & 0.39 & 0.268 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Sugar uptake (%), fermentation products concentration (g/L), biomass OD (600 nm), 2,3-butanediol yield (Y\({}_{\text{2,3-BDO/sugars}}\), g/g), and 2,3-butanediol productivity (P\({}_{\text{2,3-BDO}}\), expressed as g/L-h), at the time of maximum 2,3-butanediol production, obtained from _B. amyloliquefaciens_ grown in semi-defined media containing different initial glucose concentrations. Data in parentheses refer to sugar uptake at the end of the fermentation process (144 h). Figure 2: Fermentation kinetics in semi-defined media of glucose by _B. amyloliquefaciens_. Glucose consumption (**a**), and production of 2,3-butanediol (**b**) and acetoin (**c**). The 2,3-BDO yields achieved by _B. amyloliquefaciens_ were consistently high, ranging from 0.29 to 0.39 g/g (Table 2). Notably, the maximum yield (0.39 g/g) occurred at the highest initial glucose concentration (120 g/L). In contrast, volumetric productivity (0.265-0.348 g/L-h) peaked at 40 g/L initial glucose and decreased at higher concentrations. This decline is attributable to the longer fermentation times needed to achieve peak production under substrate stress (Table 2). Cell growth followed a similar trend to productivity. pH was also analyzed during fermentation (Figure S1b). As can be seen, independently of initial glucose concentration, pH decreased from 6.6 up to about 5.5 during the first hours of fermentation (12 h). However, after 12 h fermentation, pH increased up to 6.5-6.8 g/L (at the end of fermentation) for G20 and G40, while it decreased (up to 4.7-5.2) for G \(\geq\) 60. A key distinction from _B. licheniformis_ was the absence of ethanol production by _B. amyloliquefaciens_.