## 3 Results and Discussion  
### 2,3-BDO Production from Glucose Semi-Defined Media  
3.1.1. 2,3-BDO Fermentation by _B. licheniformis_  
To investigate the substrate and product inhibition thresholds for 2,3-BDO production by _B. licheniformis_, fermentation assays were conducted in a semi-defined medium with initial glucose concentrations ranging from 20 to 120 g/L. The results of these experiments are summarized in Table 1 and Figure 1.  
Regarding substrate consumption, _B. licheniformis_ fully metabolized the available glucose by the end of fermentation (t = 72 h) (Figure 1a). As expected, the rate of glucose consumption was inversely related to its initial concentration. For instance, in cultures initiated with 20 g/L glucose (G20), the substrate was depleted within 12 h. In contrast, complete consumption required up to 72 h in fermentations starting with 120 g/L glucose (G120). As illustrated in Table 1 and Figure 1b, final 2,3-BDO production correlated positively with the initial glucose concentration, reaching a maximum of 21.8 g/L at 120 g/L glucose. Furthermore, at initial glucose concentrations up to 80 g/L, peak 2,3-BDO production was achieved in relatively short fermentation times (t < 32 h), a finding with positive implications for process economics. Notably, high glucose uptake (>85%) occurred in all cultures by the time maximum 2,3-BDO production were reached (Table 1). While 2,3-BDO yields (from 0.12 to 0.20 g/g) and productivities (from 0.201 to 0.391 g/L-h) rose with initial glucose concentrations up to 100 g/L, concentrations exceeding this value led to a decline in both parameters (Table 1), probably due to partial inhibition of _B. licheniformis_ by high concentration of substrate and/or product. A similar trend was observed for cell growth (Table 1). pH values were also measured during fermentation (Figure S1a). As can be observed, in all cases, pH decreased from 6.6 up to about 5.5 during the first hours of fermentation (10-12 h), being increased again after this fermentation time (up to 6.9, for example, for G20 at the end of fermentation). In addition to 2,3-BDO, the fermentation yielded by-products such as ethanol and acetoin [18]. Both compounds originate from pyruvate, which is considered as an important intermediate in 2,3-BDO fermentation process; specially, ethanol is produced through successive pyruvate-formate lyase, acetaldehyde dehydrogenase, and ethanol dehydrogenase pathways; while acetoin is generated by successive \(\alpha\)-acetolactate synthase and 4,\(\alpha\)-acetolactate decarboxylase pathways [18]. While only low concentrations of ethanol were detected (\(\leq\)1.1 g/L), significant titers of acetoin (5.3-32.5 g/L) were produced across all conditions, with the maximum value observed at 120 g/L initial glucose (Table 1, Figure 1c,d). This high acetoin co-production is noteworthy given its value as  
Figure 1: Fermentation kinetics in semi-defined media of glucose by _B. licheniformis_. Glucose consumption (**a**), and production of 2,3-butanediol (**b**), acetoin (**c**) and ethanol (**d**). a platform chemical in numerous sectors, including the food, cosmetic, pharmaceutical, and agricultural industries [9]. Moreover, acetic and lactic acids can also be originated in 2,3-BDO fermentations by _B. licheniformis_[12]. In this way, notable concentrations of lactic acid (1.8-4.2 g/L) (Figure S2a) and lower amounts of acetic acid (0.1-0.6 g/L) (Figure S2b) were also detected in this work at the time of maximum 2,3-BDO production. Furthermore, it is worth mentioning that once glucose levels in the medium were depleted, _B. licheniformis_ began to consume the previously produced 2,3-BDO (Figure 1a,b). This phenomenon is directly linked to the cell's need to maintain redox balance and generate energy under nutrient-limiting conditions. The synthesis of 2,3-BDO is an NADH-dependent reduction of acetoin.