Integrating Microbial Biodiversity and Functional Potential to Assess Regulating Ecosystem Functions in a Sequential Constructed Wetland in South Korea

Constructed wetlands (CWs) are multifunctional nature-based solutions whose regulating functions are strongly mediated by microbial communities, yet microbial biodiversity is rarely integrated systematically into ecosystem-function assessment. This study operationalized a Biodiversity-Based Regulating Service Framework in a sequential CW system in South Korea using 16S rRNA gene amplicon sequencing for descriptive microbial characterization and shotgun metagenomics, an untargeted approach for sequencing community DNA, for index construction. Microbial alpha diversity, taxonomic composition, process-associated taxa, and functional genes were integrated into three Biodiversity-Based Regulating Service Indices (BBRSIs) representing pollutant removal (BBRSI-PR), nutrient retention (BBRSI-NR), and carbon-related processes (BBRSI-CS). Spatial and temporal variation was evaluated across October 2025, December 2025, and March 2026. BBRSI-PR and BBRSI-NR showed pronounced spatial differentiation in October but became higher and more uniform by March, whereas BBRSI-CS exhibited a distinct temporal pattern. BBRSI-PR and BBRSI-NR were strongly correlated (r = 0.99, p < 0.0001), while both showed weaker positive relationships with BBRSI-CS. Process-associated taxa exhibited greater spatial and temporal variability than several corresponding functional-gene groups. Overall, the framework integrated structural and functional microbial evidence into function-specific indicators capable of revealing internal heterogeneity and temporal change not apparent from inlet–outlet water-quality patterns alone.