# Distinctive Formation of Volatile Compounds in
## 2 Results and Discussion
### Volatile Compounds Based on Metabolic Pathways in Fermented Rice According to Each Microbe  
The formation of volatile compounds in fermented rice are visually represented by heatmaps in Figure 2. The fold changes of volatile compounds, which were calculated compared to the control (RICE), were classified by three main metabolic pathways, including carbohydrate, amino acid, and fatty acid metabolism (Table S1). Heatmaps were clustered by defined pathways provided from kyoto encyclopedia of genes and genomes (KEGG) pathways and previous studies [15; 16; 17]. The tendency to increase or decrease compared to the control was shown in five stages (blue; positive, red; negative, and gray; not detected), while the formation of volatile compounds which were not detected in the control but were newly produced during fermentation, were marked separately (purple; newly detected, white; not detected). As shown in Figure 2, the heatmap involved in carbohydrate metabolism was more activated compared to other forms of metabolism during rice fermentation. Carbohydrates were closely linked to the formation of some volatile compounds, such as ethanol fermentation and butanediol  
Figure 2: Metabolic pathway-based volatile compound expressions of fermented rice. The fold change of each volatile compound was calculated by comparing to control (RICE): ND; not detected, NC; not changed. Each square represents the fold change (log\({}_{2}\) level) against the control based on the color gradient as shown in the legend (blue; positive, red; negative, and gray; not detected). The formation of volatile compounds which were not detected in the control but were newly produced during fermentation were marked separately (purple; newly detected, white; not detected). Sample abbreviations are shown below; AOR (fermented by _Aspergillus oryzae_), ROR (_Rhyzopus oryzae_), SCR (_Sacharomyces cerevisiae_), SFR (_Saccharomyces fibuligera_), LFR (_Lactobacillus fermentatum_), LPR (_Lactobacillus plantarum_), and RICE (non-fermented rice). fermutation. Ethanol originates from fermentative breakdown of glucose and other hexose via the Embden-Meyerhof-Parnas (EMP) pathway and some other pathways with decarboxylase and dehydrogenase [18]. By-products of ethanol fermentation, including acetic acid, ethyl acetate, and acetaldehyde, were found in this study. In particular, acetaldehyde and acetic acid were detected only in the fermented samples, with the exception of RICE (non-fermented). The increase of acetaldehyde was higher in AOR, while that of acetic acid was superior in LPR compared to other samples. On the other hand, the overall by-products of ethanol fermentation were more actively generated in AOR, ROR, and SCR. In particular, ethanol and ethyl acetate were generated at amounts approximately 200- and 25-fold higher, respectively, than those in the control. The three-stage butanediol fermentation pathway begins with the combination of two molecules of pyruvate from glycolysis to form \(\alpha\)-acetolate by \(\alpha\)-acetolate synthase (\(\alpha\)-ALS). \(\alpha\)-Acetolate is then decarboxylated to butane-2,3-dione (diacetyl) and 3-hydroxybutan-2-one (acetoin) by \(\alpha\)-acetolate decarboxylase (\(\alpha\)-ALD) and, finally, 2,3-butanediol can be formed by the reduction of acetoin by 2,3-butanediol dehydrogenase (BDH) [19]. In this study, AOR, ROR, LFR, and LPR showed a higher generation of acetoin than other metabolites. In particular, lactic acid bacteria such as LFS and LPS were strongly associated with a higher production of acetoin, consistent with the results of a previous study [20]. On the other hand, the levels of diacetyl and acetoin in SCS and SPS were similar, while the formation of butan-2,3-diol was significantly increased in SPS. Amino acid metabolism can be involved in the generation of some volatile compounds, such as sulfur-containing volatiles, branched-chain volatiles, and aromatic volatiles [21; 22].