## 2 Results and Discussion
### VOCs Produced during Fermentation
The separation along Dim 1 was attributed to the presence of higher concentrations of \(m/z\) 43.018 (common fragment), 121.057 (t.i. benzeneacetaldehyde), 91.027 (t.i. methyl thiolacetate/mercaptoacetone), 53.006, 28.031, 42.01, 34.996, 26.016 (common fragment), 31.018, 45.033 (t.i. acetaldehyde), 27.025, 47.049 (t.i. ethanol), 83.069, 64.005, 49.011 (t.i. methanethiol), 89.060 (t.i. ethyl acetate), 81.016, 107.066 (t.i. methionol), 107.107, and 131.105 (t.i. isoamyl acetate) associated with DM samples at 7 days at 35 \({}^{\circ}\)C, in particular 43.018, 47.049, and 45.033 \(m/z\) (Figure 1b). Dim 2-explained variation was attributed to the separation of 7 days of DMfr at 25 and 35 \({}^{\circ}\)C, and DM at 25 \({}^{\circ}\)C samples from day 0 samples of DMFr at 25 and 35 \({}^{\circ}\)C, and day 7 samples of DM at 35 \({}^{\circ}\)C (Figure 1a). The separation along Dim 2 was mainly attributed to \(m/z\) 127.111, 105.081, 53.04, 107.044 (t.i. benzaldehyde), 75.043 (t.i. propanoic acid), 101.06 (t.i. 2-propenyl acetate), 73.064 (t.i. butanal), 87.08 (t.i. 2-methyl butanal and 3-methyl butanal), and 69.035 (t.i. furan) (Figure 1b). Figure 1: \(Cont.\)  
Three-way analysis of variance (ANOVA) was used to determine that of the 104 finalised \(m/z\), a total number of 66, 82, 74, 71, 55, 68, and 55 \(m/z\) were significantly (\(p<0.05\)) differentiated based upon the carbon source (medium composition), time (at 0 and 7 days), temperature (25 and 35 \({}^{\circ}\)C), medium composition \(\times\) time interactions, medium composition \(\times\) temperature interactions, time \(\times\) temperature interactions, and medium composition \(\times\) time \(\times\) temperature interactions, respectively (Table S1). Finally, 54 \(m/z\) were selected (Tables 3 and S2) for which there was a significant (\(p<0.05\)) increase in their concentration during fermentation and significant (\(p<0.05\)) differences in either main effects or interaction effects. Ethanol, which is a key marker compound in fermentation studies, is an end product of sugar fermentation by heteroformative LAB [15]. After 7 days of LB672 fermentation, the concentration of \(m/z\) 47.049 (t.i. ethanol) (Figure 2) was significantly (\(p<0.05\)) higher in DM at either 25 or 35 \({}^{\circ}\)C than in DMFr at either 25 or 35 \({}^{\circ}\)C. In DM, the concentration of ethanol was significantly (\(p<0.05\)) higher at 35 \({}^{\circ}\)C compared to 25 \({}^{\circ}\)C. When glucose is fermented by _Lev. brevis_ via the PK pathway, an extra two NAD(P)H are released during conversion of the glucose to a five-carbon sugar. The extra NAD(P)H produced is transferred to acetyl-CoA, yielding ethanol (Figure 3). In the presence of external electron acceptors like fructose, the extra NAD(P)H is transferred to the acceptors, resulting in the generation of large amounts of mannitol, which is not reduced to ethanol. When fructose is present as the only sugar, it serves both as a substrate for the PK pathway and as an electron acceptor [45], which  
Figure 1: (**a**) Score plot of the principal components (PC) of VOCs produced by LB672 across defined medium compositions (DM: glucose added (red colour), DMF: fructose added (blue colour)) at 0 (smaller size) and 7 (larger size) days of fermentation at either 25 (symbol-circle) or 35 (symbol-triangle) \({}^{\circ}\)C based on the concentrations (ppbV) of finalised 104 \(m/z\) (auto centred and scaled) from PTR-ToF-MS. (**b**) Loadings plot of the principal components (PCs) of VOCs produced by LB672 across defined medium compositions (DM: glucose added, DMF: fructose added) at 0 and 7 days of fermentation at either 25 or 35 \({}^{\circ}\)C based on the concentrations (ppbV) of finalised 104 \(m/z\) (auto centred and scaled) from PTR-ToF-MS.  
could account for the low ethanol concentration detected in DMFr. It has previously been reported that the concentration of ethanol was high in _Limosilatobacillus fermentum_ (_Lim.