## 3 Results and Discussion
### Variation of Gene Functional Profiles of the PSD and AnMBR  
Based on the above results regarding the geochemical properties and microbial communities of the PSD and AnMBR, we hypothesize that the microbial interactions and metabolic pathways of the dominant species are very different in the two bioreactors. In addition, most of the 16S rRNA gene sequences could not be classified at the family level, indicating most of the species in the PSD and AnMBR are less well-understood. Thus, comparative metagenomics was carried out to explore the divergence of the predicted microbial metabolism during methane generation in these two bioreactors. Additionally, MAGs were reconstituted to reveal the interactions of the syntrophic consortium during methane generation. Large data sets of metagenomic DNA were produced for the PSD and AnMBR samples. We generated a total of 35.9 million \(2\times 250\) bp paired-end reads, with 10.34 G and 7.27 G nucleotide bases retained after quality filtering for the PSD and AnMBR samples, respectively. The metabolic potential of the microbial communities of the PSD and AnMBR was predicted by comparison to the KEGG Ortholog (KO) database. In the PSD and AnMBR metagenomes, genes encoding metabolic functions were dominant, representing 20.22% and 22.79% within each sample, respectively, followed by genetic information processing (4.56% in the PSD and 5.07% in the AnMBR) and environmental information processing (3.30% in the PSD and 4.25% in the AnMBR) (Figure S2). In the category of metabolism, the most abundant metabolic type was carbohydrate metabolism (4.28% in the PSD and 4.82% in the AnMBR), followed by amino acid metabolism (4.19% in the PSD and 4.73% in the AnMBR) as well as energy metabolism (3.15% in the PSD and 3.35% in the AnMBR) (Figure S2). A high proportion of genes related to these functions has been previously reported in metagenomes [49; 50; 51], metotranscriptomes [52] and metaproteomes [53] from AD systems, indicating these metabolic activities are linked to the conversion of excess carbohydrates into methane during anaerobic digestion. When classified as KEGG level 3, the abundance of genes in methane metabolism was one of the categories with the highest abundance in the PSD and AnMBR, consisting of 1.05%  
Figure 2: Taxonomic composition of the microbial communities showing the differences between the PSD and AnMBR samples. The data reveal the phylum- and genus-level classification of the 16S rRNA gene sequences. 16S rRNA gene sequences longer than 250 bp were classified using the RDP naive Bayesian rRNA Classifier at an 80% confidence threshold. Only the relative abundances of the identified phyla higher than 1% are listed. The average \(\pm\) SD values of the samples in each group are expressed in each column. The significance of the cutoff for the corrected \(p\)-value using the Benjamini–Hochberg FDR procedure was set at 0.05. The significant differences within the sampling group are labeled **, where \(p<0.05\). and 0.88% of the total sequences (Table S5), respectively, suggesting methanogenesis was the major microbial metabolism in these two bioreactors. To better understand the difference in carbohydrate decomposition in the PSD and AnMBR, we screened our metagenome for reads annotated as CAZyme. The PSD metagenome contained 864,945 reads (consisted of 4.17% of the total reads) associated with CAZyme that were unequally distributed among glycosyltransferases (GTs), glycoside hydrolases (GHs), carbohydrate binding modules (CBMs), carbohydrate esterases (CEs), auxiliary activities (AAs), and polysaccharide (PLs) (Table S6), and their read abundances were 41.3%, 37.2%, 12.8%, 5.3%, 2.6%, and 0.8%, respectively (Figure S3). In the AnMBR metagenome, the read abundances of GIs, GHs, CBMs, CEs, AAs, and PLs were 43.4%, 37.8%, 12.0%, 4.4%, 2.6%, and 0.8%, respectively (Figure S3). A higher gene abundance of CAZyme suggests a greater role of carbohydrate metabolism in the anaerobic digestion of the PSD and AnMBR.