Enhancing aerobic granular sludge formation and pollutant removal via bioaugmentation with indigenous EPS-producing...

Aerobic granular sludge (AGS) has emerged as a promising biological wastewater treatment technology due to its compact reactor footprint, excellent settleability, and ability to simultaneously remove carbon, nitrogen, and phosphorus within a single sequencing batch reactor (SBR). However, its practical application remains limited by the prolonged granulation period during reactor start-up and the instability of mature granules under continuous operation. This study investigated the use of indigenous extracellular polymeric substance (EPS)-producing bacteria as a bioaugmentation strategy to accelerate granule formation and improve reactor performance. Two laboratory-scale SBRs with a working volume of 2.5 L were operated using real domestic wastewater collected from the Bunus Sewage Treatment Plant, Malaysia, under 3-h operating cycles. Indigenous bacterial isolates obtained from activated sludge were screened based on biofilm formation, fluorescence spectroscopy, and aggregation ability. Two strains, Bacillus albus and Proteus mirabilis, were selected for co-inoculation because of their complementary functional characteristics, namely high surface adhesion and strong protein-like EPS fluorescence. The bioaugmented reactor achieved 50% granulation (dry mass >0.2 mm) within 10 days and complete conversion of flocs into granules by day 40, whereas the control reached only 5% and 80% granulation at the same time points. By day 10, the bioaugmented sludge reached a settling velocity of 2.24 m h^-1 (vs. 1.37 m h^-1 in the control), and its SVI₅ decreased to 76 mL g^-1 by day 40 (vs. 108 mL g^-1). FE-SEM/EDS analysis revealed a dense EPS matrix with higher carbon and silicon contents than the control. The bioaugmented reactor consistently achieved higher pollutant removal efficiencies, reaching nearly 100% for COD, 75-90% for NH₃-N, 70-83% for NO₃^--N, and about 60% for TP. 16S rRNA gene amplicon sequencing further showed enrichment of Nitrospira (0.95% to 9.94%), Candidatus Accumulibacter (0.13% to 1.83%) and Chloroflexi, whereas the inoculated genera were not detected in the mature granules, suggesting that they acted mainly during early aggregation. Overall, the findings demonstrate that bioaugmentation with indigenous EPS-producing bacteria is an effective approach for accelerating AGS start-up and improving granule stability and treatment performance under tropical domestic wastewater conditions.