## 3 Results and Discussion
### 2,3-BDO Production from Glucose Semi-Defined Media
#### 3.1.3 Comparison of 2,3-BDO Fermentation by _B. licheniformis_ and _B. amyloliquefaciens_  
A direct comparison of the two strains revealed key differences in substrate utilization and fermentation kinetics. _B. licheniformis_ achieved complete glucose consumption across all tested initial concentrations (20-120 g/L), whereas _B. amyloliquefaciens_ demonstrated efficient uptake (\(\geq\)90%) only at concentrations up to 80 g/L (Tables 1 and 2, Figures 1 and 2). Furthermore, _B. licheniformis_ exhibited faster overall kinetics, completing fermentation in 72 h compared to 144 h for _B. amyloliquefaciens_ (Figures 1a and 2a). At initial glucose concentrations between 20 and 80 g/L, _B. amyloliquefaciens_ produced significantly higher 2,3-BDO titers (_p_ \(<\) 0.05), yielding 1.4- to 2.4-fold more product than _B. licheniformis_ (Tables 1 and 2, Figures 1b and 2b). However, this advantage was lost at concentrations above 80 g/L. At 100 g/L initial glucose, the 2,3-BDO production from both strains was comparable (18.0-19.8 g/L), while at 120 g/L, _B. amyloliquefaciens_ produced substantially less 2,3-BDO than _B. licheniformis_ (15.0 g/L vs. 21.8 g/L). These results indicate that _B. amyloliquefaciens_ is more susceptible to substrate inhibition at high glucose loads (>80 g/L) than _B. licheniformis_. Regarding process efficiency, _B. amyloliquefaciens_ demonstrated significantly higher 2,3-BDO yields (_p_ \(<\) 0.05), ranging from 0.29 to 0.39 g/g compared to 0.12-0.20 g/g for _B. licheniformis_ (Tables 1 and 2). The yield from _B. amyloliquefaciens_ reached up to 78% of the theoretical maximum (0.5 g/g) [23]. In contrast, _B. licheniformis_ consistently reached its peak titer more rapidly (e.g., t = 28 h vs. 56 h at 60 g/L glucose). Consequently, despite the yield disparity, the overall volumetric productivities of the two strains were not significantly different (_p_ \(>\) 0.05), with ranges of 0.201-0.391 g/L-h for _B. licheniformis_ and 0.265-0.348 g/L-h for _B. amyloliquefaciens_. Similarly, final cell densities were comparable between the strains (OD\({}_{600}\) 6.5-16.2 vs. 8.0-14.4) (Tables 1 and 2). Considering pH parameter, although it decreased during the first 10-12 h of fermentation for both microorganisms, after this fermentation time, for G \(\geq\) 60 g/L initial glucose, _B. licheniformis_ increased the pH of medium, while a decrease in pH was observed in fermentations by _B. amyloliquefaciens_ (Figure S1a,b). The two strains also exhibited distinct by-product profiles. _B. licheniformis_ was a more prolific producer of acetoin, generating significantly higher concentrations (5.3-32.5 g/L) than _B. amyloliquefaciens_ (3.3-10.3 g/L) (Tables 1 and 2, Figures 1c and 2c). Crucially, the production dynamics differed: _B. licheniformis_ co-produced acetoin and 2,3-BDO from the onset of fermentation, whereas acetoin synthesis by _B. amyloliquefaciens_ was delayed, increasing only as glucose was depleted and 2,3-BDO levels rose. Consistent with our results, _B. licheniformis_ has been previously recognized for its capacity to co-produce significant amounts of acetoin alongside 2,3-BDO [21]. In contrast, various _B. amyloliquefaciens_ strains are often cited for their high efficiency in selectively producing 2,3-BDO. In this way, for example, [PERSON] et al. [22] reported that _B. amyloliquefaciens_ produced high titers of 2,3-BDO (20.6-40.7 g/L) from spirit-based distilles' grain, with comparatively low acetoin co-production (2.9-5.4 g/L). This metabolic bifurcation towards either acetoin or 2,3-BDO is regulated by several factors, most notably the intracellular redox state (NAD\({}^{+}\)/NADH ratio) and the concentration of dissolved oxygen in the medium [24]. The final conversion step, the reduction of acetoin to 2,3-BDO, is NADH-dependent. Therefore, the direction of this reversible reaction is highly sensitive to environmental conditions.