ADVANCED MOLECULAR TOOLS FOR WASTEWATER TREATMENT OPTIMIZATION WITH FOCUS ON SLUDGE BULKING

Abstract

Effective microbial population management is critical to the performance of wastewater treatment operations, particularly in addressing filamentous bulking, which compromises sludge settling and effluent quality. Traditional bulking indicators, such as the sludge volume index (SVI), and microscopic identification of filamentous bacteria is typically performed only after bulking has occurred. This highlights the need for early-warning indicators of bulking events. The present study investigates the application of advanced molecular tools within a full-scale wastewater treatment context, evaluating whether these techniques offer advantages over traditional monitoring methods in managing activated sludge systems. Consequently, a full-scale evaluation was initiated to assess the predictive value of the specific floc-bulking Adenosine Triphosphate (s-fbATP) and to investigate the microbial basis of bulking. Initial system-wide assessments revealed clear ecological differentiation between two intermittently aerated tanks, which operated in either online (active) or offline (inactive) modes depending on seasonal conditions and hydraulic load. Principal coordinates analysis (PCoA) showed that microbial communities in Aeration Tank 3 (AT3) were tightly clustered, indicating ecological stability, whereas communities in Aeration Tank 2 (AT2) exhibited marked separation between active and inactive operational states. Multivariate analysis of variance (MANOVA) confirmed a significant tank-state interaction in AT2 but not in AT3. Based on these findings, AT3 was selected for detailed microbial and operational analysis. Time-series analysis over an 18-month period indicated that increases in s-fbATP consistently preceded rises in SVI by approximately 7 days, with the strongest cross-correlation (r = 0.67) observed when s-fbATP levels exceeded the 50% threshold, indicating conditions that required operational intervention. These elevated s-fbATP periods were frequently characterized by declining effluent quality, as indicated by increased BOD (22.0 ± 6.8 mg/L) and COD (129.9 ± 1.9 mg/L). Conversely, when s-fbATP values remained below the 30% interrupt ratio, effluent quality improved, with average BOD and COD levels of 7.0 ± 1.7 mg/L and 94.4 ± 20.4 mg/L, respectively. A filtration-based validation test confirmed that the DNA retained on a 250 µm filter used as part of the fbATP test was in fact enriched in filamentous and variable filamentous bacteria. Principal component analysis (PCA) revealed distinct community separation between the filter-retained fraction and the filtrate, supporting the analytical specificity of the fbATP assay for filamentous biomass, but additional verification in more locations and conditions is needed. Episodes of bulking were statistically associated (p < 0.05) with elevated levels of organic loading (COD, F/M), ammonia, sulfate, and chloride. 16S rRNA gene sequencing and Thiothrix-specific quantitative PCR identified Thiothrix as the dominant filamentous genus; however, its abundance explained only a small fraction of the variation in s-fbATP and SVI. Ridge regression and microbial community analyses suggested that multiple taxa were associated with bulking events. Notably, increases in filamentous genera classified as Mycobacterium, Flavobacterium, and Acinetobacter showed positive correlations with chromium and nickel concentrations (r = 0.72), these findings are based on limited samples. While Thiothrix abundance showed a positive correlation with sulfate (r = 0.51) and higher pH; it was negatively associated with cadmium, lithium, titanium, and selenium. Overall, the data indicate that s-fbATP showed promise as an operational metric and a indicated a potential one-week early-warning signal for sludge bulking and effluent quality deterioration., which would require more widespread validation. The results also suggest that filamentous growth is primarily driven by community-level responses, involving multiple microbial taxa rather than a single pathogenic genus. Routine monitoring of s-fbATP, particularly when integrated with molecular profiling techniques, could enhance early detection and support more proactive management of sludge settleability and effluent quality in biological wastewater treatment systems.