Substrate-Driven Microbiome Assembly in Water Hyacinth Vermicompost: Combined 16S rRNA and Shotgun Metagenomics for Sustainable Agriculture.

Substrate composition is a primary determinant of microbial succession and functional dynamics in vermicomposting systems. However, comparative insights into how biomass pre-treatment influences microbial architecture and how different sequencing approaches capture these changes remain limited. In this study, evaluation was carried out on microbial community structure and metabolic potential in vermicompost derived from three forms of Eichhornia crassipes (water hyacinth) biomass, burnt biomass (BB), composted biomass (CB) and dry biomass (DB) using both 16S rRNA gene amplicon sequencing and shotgun metagenomics. All treatments were dominated by bacterial communities (> 97%), with Proteobacteria (Pseudomonadota), Firmicutes (Bacillota), Actinobacteria and Bacteroidota representing core phyla across substrates. However, metagenomics revealed broader domain-level coverage, detecting Archaea and Fungi that were underrepresented in 16S datasets. Substrate-specific signatures were evident such as, composted biomass exhibited enrichment of lignin degradation and carbon cycling pathways; dry biomass showed methanogenesis, fermentation and phosphate solubilization signatures; and burnt biomass was associated with nitrogen fixation and sulphur metabolism. Shannon diversity was highest in composted biomass (H' = 5.21), reflecting enhanced niche diversification during substrate maturation. Comparative analysis demonstrated that 16S rRNA sequencing effectively captured dominant bacterial structure, whereas shotgun metagenomics provided superior taxonomic resolution and direct functional inference, particularly for low-abundance and non-bacterial taxa. Notably, functional differentiation among treatments was more pronounced than broad taxonomic shifts, indicating that biomass pre-treatment exerts stronger influence on ecological function than on core community composition. These findings demonstrate that integrating taxonomic and functional metagenomics enables substrate-specific optimization of vermicompost formulations and provides a framework for designing microbiome-informed strategies for sustainable agriculture and invasive biomass valorization.