Unlocking microbial synergy in microalgae-bacteria granules: RSM-driven optimization and lifecycle microbial...

Landfill leachate is challenging to treat biologically due to high ammonia toxicity and low C/N ratio, while suspended co-cultures, despite easing carbon limitations, show poor settleability and instability under high-strength conditions. This work focuses on investigating microbial synergy in microalgae-bacteria granules through RSM-driven optimization and lifecycle microbial metagenomics for high-ammonia leachate treatment. Initially, granules removed Total inorganic nitrogen (TIN) at 167.5 mg/L/d, outperforming algae-only systems. 16S rRNA analysis showed 25 % more Halomonas sp. and enriched Proteobacteria, linked to improved pollutant removal. Subsequent RSM optimization elevated TIN removal to 193.3 mg/L/d, with synchronous improvement in chemical oxygen demand (COD) removal, flocculation, Extracellular Polymeric Substances (EPS) production, and granule stability. In addition, reactor operation over 8 cycles sustained TIN removal above 193.4 mg/L/d until Cycle IV and then dropped by 5.0 %, displaying a typica granule life cycle from growth to decline. Metagenomic analysis of 326 metagenome-assembled genomes (MAGs) revealed dynamic microbial synergy that shifted from nutrient assimilation to stress adaptation, explaining the system's resilience. During assimilation-dominated stages, synergy focused on coordinated nutrient utilization, supporting energy-intensive nitrogen assimilation. As metabolism shifted to dissimilation, synergy reoriented toward stress adaptation: assimilation genes decreased to 44.3 % of their peak levels, while stress genes hcp and cah increased by 11.2-fold. This dynamic synergy provides a blueprint for lifecycle management. These findings highlight the great potential of microalgae-bacteria granules as a resilient, low-footprint approach for nitrogen removal and circular wastewater treatment.