## 3 Results
### Changes in Bacterial Community Composition  
The sequences with more than 0.1% valid sequences in the 12 EFB samples were annotated. At the phylum level, the bacterial community in the EFB was mainly composed of _Firmicutes_, and _Bacteroidetes_ (10.93%) was found only in sample P13-1 (Figure 3A). In addition, the sequence alignment of 58 ASVs obtained from the 12 samples and whose proportion was greater than 0.1% showed that 44 of the ASVs had a similarity \(\geq 97\%\) with the sequences of the culturable microorganisms available on the NCBI database, whereas 14 of the ASVs had a similarity \(<97\%\) with these sequences NCBI, thus indicating that there were potentially new species in the EFB. Furthermore, we merged the ASVs with a sequence similarity of \(\geq 97\%\) into the same OTU, and the fifty-eight ASVs were grouped into twenty-nine OTUs belonging to nine genera, which were assigned to three families, including twenty-one known species and eight unclassified species (Figure 3B and Table S2). Twelve representative ASVs, ASV134, ASV95, ASV104, ASV135, ASV109, ASV103, ASV196, ASV136, ASV152, ASV137, ASV101, and ASV96, were the dominant species, which accounted for 61.39-95.3% of the samples. These dominant ASVs were mainly composed of _Clostridium_, _Caproicibacterium_, _Pediococcus_, _Lacticaseibacillus_, _Weizmannia_, and potential novel species within the order Oscillatoriaceae (Figure 3B and Table S2). Figure 3: Distribution characteristics of bacterial community structures of samples in different enrichment rounds. (**A**) Percentage of community abundance at the phylum level. (**B**) Percentage of community abundance at the ASV level (the ASV was compared with the NCBI database, the species with a similarity of greater than 97% were grouped, and potential new species were classified and merged. Compared with the database, the similarity between ASV sequences is less than 97%, and the ASV sequences with similarity higher than 97% were classified as new species). In addition, in order to better understand the composition and succession patterns of the dominant microbial communities in each enrichment round (abundance \(\geq\) 1% in one of 1-6 rounds and more dominant than other rounds), we inversed the community-successional pattern in each sample at the species level based on the ASV-alignment data (Figure 3B). In the first round of the enrichment, samples P13-1 and P14-1 were greatly affected by the PM and heat-shock treatment, which resulted in significant differences in microbial composition. Comparing the differences in microbial composition between samples P13-1 and P14-1, it was possible to determine which microorganisms are sensitive to heat shock. Specifically, the dominant species in the P13-1 were _Clostridium neuense_ (45.46%), _Clostridium guangxiense_ (10.32%), _Clostridium diolis_ (6.31%), _Lactobacillus acetolorans_ (5.45%), _Clostridium liquoris_ (4.52%), _Clostridium algifieecis_ (3.89%), _Clostridium fernenticellae_ (3.33%), _Clostridium kogasense_ (3.26%), _Clostridium luticellari_ (2.24%), _Clostridium tepidum_ (1.08%), and potential new species found within the orders _Bacteroidales_ and _Oscillospiraceae_, accounting for 10.93% and 1.27% of the abundance, respectively. In contrast, the dominant microorganisms in the P14-1 were _C. guangxiense_ (58.00%), _Weizmannia coagulans_ (34.62%), _C. fermenticellae_ (3.10%), _C. luticellarii_ (1.65%), and _C. kogasense_ (1.08%). By comparing the differences in microbial community composition between samples P13-1 and P14-1, it was revealed that the heat-shock treatment inhibited several species within the genera _Clostridium_ and _Lactobacillus_, as well as within the order _Bacteroidales_, which are probably thermolabile microorganisms, or organisms not adapted to CGM, except for _C. guangxiense_, _C. fermenticelles_, _C. luticellarii_, and _C. kogasense_, which occurred simultaneously in samples of the two treatments.