To examine the relationship between activated-sludge bulking and levels of specific filamentous bacteria, we developed a statistics-based quantification method for estimating the biomass levels of specific filaments using 16S rRNA-targeted fluorescent in situ hybridization (FISH) probes. The results of quantitative FISH for the filament Sphaerotilus natans were similar to the results of quantitative membrane hybridization in a sample from a full-scale wastewater treatment plant. Laboratory-scale reactors were operated under different flow conditions to develop bulking and nonbulking sludge and were bioaugmented with S. natans cells to stimulate bulking. Instead of S. natans, the filament Eikelboom type 1851 became dominant in the reactors. Levels of type 1851 filaments extending out of the flocs correlated strongly with the sludge volume index, and extended filament lengths of approximately 6 x 10(8) micro m ml(-1) resulted in bulking in laboratory-scale and full-scale activated-sludge samples. Quantitative FISH showed that high levels of filaments occurred inside the flocs in nonbulking sludge, supporting the "substrate diffusion limitation" hypothesis for bulking. The approach will allow the monitoring of incremental improvements in bulking control methods and the delineation of the operational conditions that lead to bulking due to specific filaments.

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Activated sludge bulking is normally considered to occur when the sludge volume index (SVI) reaches 150 mL/g. Previous work has showed that above a certain level of total extended filament length (TEFL), the SVI increases rapidly. This filament length level can thus be considered the threshold for bulking to occur due to all the filamentous bacteria present. In this study, the quantitative FISH method was combined with lab-scale reactor experiments to measure threshold values of filament length for bulking caused by a specific filamentous species. Three lab-scale reactors were operated to allow tracking of filaments before and during the bulking episodes: one sequencing batch reactor (SBR) with fast feeding (Reactor A); one SBR with slow feeding (Reactor B); and one completely mixed reactor with continuous feeding (CMR, Reactor C). One week after the addition of S. natans, Reactor C suddenly experienced bulking, and the SVI increased from 92.9 mL/g to 198.2 mL/g in one day. Reactor B began bulking after 4 more days. The SVI for Reactor A remained constant at 40 mL/g. Interestingly, fluorescence in situ hybridization (FISH) analysis showed that instead of S. natans, Eikelboom Type 1851 was the dominant filament in the sludge during the whole course of bulking in Reactors B and C. The quantitative FISH showed that SVI and filament length of Type 1851 strongly correlated with each other (r=0.98), which suggests that Type 1851 proliferation may be the cause of bulking in reactor C. The threshold level for the onset of bulking occurs at 8 x 10 8 µm/mL total filament length, or around 8 x 10 8 µm/mL extended filament length.