## 2 Materials and Methods
### Bacterial Community Sequencing Analysis  
After demultiplexing, the resulting sequences were quality-filtered with fastp (0.19.6) [23] and merged with FLASH (v1.2.11) [24]. At that point the high-quality groupings were de-noised utilizing the DADA2 [23] plugin within the Qime2 [25] (adaptation 2020.2) pipeline with prescribed parameters, which obtains a single-nucleotide determination based on error profiles within samples. DADA2-denoised arrangements are ordinarily called amplicon sequence variants (ASVs). To reduce the impact of the sequencing on the alpha- and beta-diversity degree, the number of groupings from each test was rarefied to 20,000, which still yielded a normal Good's scope of 99.95%. Taxonomic assignment of ASVs was performed utilizing the Naive Bayes consensus scientific classification classifier actualized in Qime2 and the SILVA 16S rRNA database (v138). The metagenomic work was used PICRUST2 (Phylogenetic Examination of Communities by Reproduction of Imperceptibly States) [26] based on ASV agent groupings. PICRUST2 is a computer program containing an arrangement of tools as follows: HMMER was utilized to adjust the ASV agent arrangements with reference groupings. EPA-NG and Gappa were utilized to put the ASV agent arrangements into a reference tree. The castor was utilized to normalize the 16S quality duplicates. MinPath was utilized to predict quality family profiles, and find the quality pathways. Entire analysis process was accord to protocols of PICRUST2.  
A bioinformatic analysis of the silage microorganism was carried out using the Major-bio Cloud platform ([[https://cloud.majorbio.com](https://cloud.majorbio.com)]([https://cloud.majorbio.com](https://cloud.majorbio.com)) (accessed on 1 December 2024)). Based on the ASV information, rarefaction curves and alpha diversity indices including observed ASVs, Chao1 richness, Shannon index, and Good's coverage were calculated with Mothur v1.30.1 [27]. The similarity among the microbial communities in different samples was determined by principal coordinate analysis (PCoA) based on Bray-Curtis dissimilarity using the Vegan v2.5-3 package. The PERMANOVA test was used to assess the percentage of variation explained by the treatment along with its statistical significance using the Vegan v2.5-3 package. The linear discriminant analysis (LDA) effect size (LEfSe) [28] ([[http://huttenhowersph.harvard.edu/LEfSe](http://huttenhowersph.harvard.edu/LEfSe)]([http://huttenhowersph.harvard.edu/LEfSe](http://huttenhowersph.harvard.edu/LEfSe)) (accessed on 1 December 2024)) was performed to identify the significantly abundant taxa (phylum to genera) of bacteria among the different groups (LDA score > 2, \(p\) < 0.05). A distance-based redundancy analysis (db-RDA) was performed using the Vegan v2.5-3 package to investigate the effect of nutritional quality and fermentation quality on the silage bacterial community structure. The forward selection was based on Monte Carlo permutation tests (permutations = 9999). The values of the x- and y-axes and the length of the corresponding arrows represent the importance of each nutritional quality and fermentation quality in explaining the distribution of taxon across communities. Linear regression analysis was applied to determine the association between major nutritional quality and fermentation quality, as identified by db-RDA analysis and microbial alpha diversity indices.