Anaerobic digestion (AD) of thickened waste-activated sludge (TWAS) is widely applied for sludge stabilization and renewable energy recovery; however, hydrolysis of complex organics often limits fermentation performance. This study evaluated the effects of multiple pretreatment strategies on solubilization, volatile fatty acids (VFAs) production, and extracellular polymeric substances (EPS) during 80 h mesophilic batch fermentation. Pretreatments included hydrothermal treatment (HTP; 70, 90, and 170 °C), ultrasonication (US; 3000, 5000, and 10,000 KJ/kg TS), chemical pretreatment (acidic pH 4 and alkaline pH 10), and biological augmentation using YDRO Process^® (YDRO^®; 5%, 10%, 15% v/v). Across feedstock pretreatments, HTP generated the greatest improvements in solubilization, increasing SCOD by 56–113-fold and producing substantial acetate levels, particularly at 70 °C, alongside substantial phosphorus release. Ultrasonication resulted in moderate solubilization (28–56-fold) and elevated soluble phosphorus and ammonia. Acidic pretreatment maximized soluble phosphorus, but showed limited VFAs production, whereas alkaline pretreatment rapidly increased soluble EPS due to pH-induced cell disruption. Bioaugmentation achieved the highest total COD but yielded comparatively low soluble fractions. Following fermentation, HTP 170 °C consistently outperformed other treatments, maintaining elevated soluble COD and producing the highest acetate concentration. EPS analysis revealed extensive protein and polysaccharide degradation in thermal and bioaugmented systems, indicating active utilization during fermentation. Overall, the results demonstrate that targeted pretreatment strategies significantly enhance organic solubilization, EPS disruption, and VFAs yields, with thermal pretreatment showing the greatest potential to accelerate hydrolysis and acidogenesis. These findings provide valuable insights for optimizing the pre-methanogenic stages of AD and improving the efficiency of sludge treatment and resource recovery.

Extracellular polymeric substances (EPS) play an important role in wastewater sludge structure and stability, yet measured EPS concentrations depend strongly on the extraction method. This study compared three established protocols (thermal sodium carbonate, thermal sodium chloride, and chemical formamide–NaOH extraction) across activated sludge (AS), aerobic granular sludge (AGS), algal–bacterial granular sludge (ABGS), and an AGS–membrane bioreactor (AGS-MBR). Proteins (PN) and polysaccharides (PS) were quantified in soluble, loosely bound, and tightly bound EPS fractions, with total EPS defined as PN + PS. Sodium carbonate consistently produced the highest apparent PN, PS, and total EPS recoveries across the sludge matrices, while sodium chloride and chemical extraction generally produced lower and comparable recoveries. Because the protocols differed in reagent chemistry, temperature, incubation time, and centrifugation conditions, these differences cannot be attributed to a single extraction parameter. A complementary Escherichia coli experiment showed that adding intact cells to an autoclaved-sludge background altered subsequent polymer recovery during sodium carbonate extraction, suggesting a potential contribution of cellular-associated material; however, PN and PS measurements cannot directly demonstrate cell lysis. Overall, extraction-method selection should consider both recovery performance and potential extraction-induced cellular contributions when interpreting and comparing EPS measurements.

Efficient simultaneous nitrification, denitrification, and phosphorus removal typically requires either separate tanks with oxic and anoxic conditions or aerobic granular sludge (AGS) with granules larger than 0.2 mm. In this study, small granular flocs (50–100 µm) were cultivated with improved settleability, high biomass retention, strong ammonia and COD removal, late-stage phosphate removal, and enrichment of Paracoccus. The sequencing batch reactor was seeded with biomass from a municipal wastewater treatment plant and fed acetate-based synthetic wastewater for 114 days. The small granular flocs formed densified activated sludge (DAS), with biomass accumulating to approximately 12 g/L mixed liquor suspended solids. DAS developed under selection conditions commonly applied in aerobic granular sludge systems, including unaerated feeding and mixing, elevated hydrodynamic shear, and progressive reduction in settling time. The sludge volume index (SVI) improved to 30 mL/g, comparable to that of granular sludge, with a moderate SVI₃₀/SVI₅ ratio of 72%. The enrichment of Paracoccus is highly desirable in wastewater treatment. Importantly, these findings show that efficient simultaneous nutrient removal does not strictly depend on large granular structures.

Efficient simultaneous nitrification, denitrification, and phosphorus removal typically requires either separate tanks with oxic and anoxic conditions or aerobic granular sludge (AGS) with granules larger than 0.2 mm. In this study, we cultivated small granular flocs (50-100 µm) capable of simultaneous heterotrophic nitrification and aerobic denitrification (HN-AD) in a sequencing batch reactor. The bioreactor was seeded with biomass from a municipal wastewater treatment plant and fed acetate-based synthetic wastewater for 114 days. The small granular flocs formed densified activated sludge (DAS), with biomass accumulating to approximately 12 g/L mixed liquor suspended solids. DAS was cultivated under selection pressures commonly used for AGS formation, including anoxic feeding, extended anoxic mixing, elevated hydrodynamic shear, and a gradual reduction in settling time. The sludge volume index (SVI) improved to 30 mL/g, comparable to that of granular sludge, with a moderate SVI₃₀/SVI₅ ratio of 72%. The relative abundance of Paracoccus reached 72.9%, indicating the metabolic capability of these small granular flocs to carry out HN-AD. Overall, these findings show that efficient simultaneous nutrient removal does not strictly depend on large granular structures. Achieving DAS and HN-AD offers a scalable approach to intensify biological nutrient removal while reducing reliance on distinct anoxic zones.