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

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 SVI 5 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 NH 3 –N, 70–83% for NO 3 − –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.