## 4 Discussion
### Correlation between Dominant Acid-Producing Microorganisms and Organic Acid Contents
pabulibutyricum_ (ASV136), which can produce acetic acid, butyric acid, or mixed acid, showed a positive correlation with the contents of acetic acid and butyric acid in the EFB samples. Lactic acid is a non-volatile acid that cannot be detected by GC-MS. Although ethyl lactate synthesized from lactic acid is an important volatile compound in SFB, relevant studies suggested that the excessive production of lactic acid inhibits the formation of the main flavor (ethyl caproate) of SFB [61]. In this study, _L. casei, Levilobacillus brevis, P. acidilactici, W. coagulans_, and _Secundiobacillus mixtipabul_ were enriched. These microbial groups (L_a) were negatively correlated with acetic acid, butyric acid, and caproic acid. Relevant studies also reported that, when found in high abundance, lactic acid bacteria can inhibit clostridia and caproic acid production by lowering environmental pH, which leads to the accumulation of lactic acid and/or the secretion of some bacterioxins [12]. In addition, it was found that the potentially dominant communities (ASV 142, ASV 103, ASV 116, ASV 119, ASV 153, ASV 145, and ASV 148) newly identified in the FEB samples were also positively correlated with the caproic acid content. Among these, the ASV 142, ASV 103, ASV 116, and ASV 119 shared 94.55-96.53% similarity with _C. amylolyticum_, whereas the ASV 153, ASV 145, and ASV 148 shared 93.87-94.85% similarity with _C. galactilovirans_; therefore, it can be speculated that these may potentially constitute novel species with caproic-acid-production ability. In addition, the ASV135 and ASV190 were positively correlated with the acetic acid and butyric acid contents. The ASV135 and ASV190 shared 92.89-93.38% similarity with the _16S rRNA_ gene sequence of _O. ruminantium_ GH1. Related studies also showed that _O. ruminantium_ GH1 can use D-glucose, D-ribose, and D-xylose to produce butyric acid [26]. Therefore, it can be speculated that ASV135 and ASV190 may have the ability to produce butyric acid. Interestingly, the potential novel species _Bacteroidetes_ was found only in the P13-1 samples, which were negatively correlated with the contents of acetic acid, butyric acid, and caproic acid. Thus, these species may have other functions. Analysis of Community-Succession Pattern and Enrichment Stage of Specific Acid-Producing Microorganisms  
During our multi-round enrichment fermentation, the microbial community compositions in samples P13-1 and P14-1 significantly differed due to the application of heat-shock  
Figure 4: Relationship between microbial community and organic acid composition. (**A**) CCA analysis of functional microorganisms and organic acids. (**B**) Spearman analysis of functional microorganisms and metabolites (Aa: acetic acid; Ba: butyric acid; Ca: caproic acid; C_a: microorganisms capable of producing caproic acid; AB_a: microorganisms capable of producing acetic acid, butyric acid, or mixed acid; L_a: microorganisms capable of producing lactic acid). (more significant difference: *\(p\leq 0.05\); significant difference: **\(p\leq 0.01\)). treatment in round 1 of the enrichment in samples P14-1 (Figure 3B). Relevant studies found that heat treatment can reduce the abundance of certain non-spore-forming microorganisms, such as _Lactobacillus_, a few clostridia, and some organisms within the genus _Bacteroides_ (e.g., _Bacteroides_, _Odoribacter_, and _Alloprevotella_) [62; 63]. Comparing the microbial community structures in rounds 2-6 of the enrichment, it was shown that the CGM had strong selectivity for the anaerobic microorganisms in the PM. Among these, the dominant microbiota capable of metabolizing volatile acids in samples P13-2 and P14-2 during the adaptation stage of the enrichment were _C. kogasense_, _C. liquoris_, and _C. fermenticellae_, as well as ASV116 and ASV119 (Figures 5A and 4B). In the main fermentation stage, the abundance of _C. kogasense_ started to decrease, while those of _C. fermenticellae_, _C. amylolyticum_, _C. guangxiense_, _C. algrifiecis_, _C.