Adhesins: The missing link for activated sludge bioflocculation

Activated sludge bioaggregation is critical to the proper performance of biological wastewater treatment systems because without an efficient sludge-water separation, the effluent quality deteriorates, and slow-growing organisms might wash out. Activated sludge flocs are mainly composed of floc-forming and filamentous bacteria and extracellular polymeric substances. Among the extracellular polymeric substances are the amyloid adhesins: highly stable fibrillar molecules with a characteristic β-sheet structure. These molecules are thought to contribute to bioaggregation. The overall aim of this work is to make process optimization in biological wastewater treatment more targeted by understanding the bioaggregation mechanisms and, in particular, the role of amyloid adhesins. Therefore, the impact of amyloids on flocculation performance, and the variety of amyloid producers and amyloid proteins was investigated. Thioflavin T, for which the staining procedure was first critically assessed, was used to fluorescently detect amyloid-like substances in sludge.

The abundance and distribution of amyloids and their potential producers were monitored in two lab-scale reactor experiments. The reactors were operated in sequencing batch mode with an unaerated and aerated reaction phase and either feast-famine or more continuous feeding was applied. Thioflavin T staining revealed more amyloid-like substances in the lab-scale reactors during all operational stages than in the inocula. The morphology of thioflavinophilic areas in the sludge was diverse (clustered within microcolonies, diffuse throughout the floc, or on single cells
or filaments), suggesting a high diversity of producers and functions. Sequential Thioflavin T staining and fluorescence in situ hybridization identified Zoogloea and ‘Candidatus Competibacter’ as potential amyloid producers under the conditions used.

However, in this mixed-culture approach, there are simultaneous dynamic interactions between the operational conditions, the bacterial community, the composition and quantity of the extracellular material, and the floc and sludge properties. As a result, it was not possible to identify specific operating conditions to affect amyloid production in a targeted way and to investigate the direct relationship between amyloid production and aggregation. Therefore, the first steps were taken towards an alternative experimental approach based on pure cultures of amyloid producers. Dietzia maris and Azoarcus communis were identified as potential amyloid producing strains, although the specific identity of their thioflavinic compounds remains to be determined.

As an application, the augmentation of sludge with structural extracellular polymeric substances producers was explored as a bioflocculation improvement or remediation approach. An antibiotic resistant and fluorescent-protein producing mutant of Azoarcus communis was constructed to monitor its survival and location in a microbial community in a membrane bioreactor. Long-term survival of the organisms was limited so the effect on bioflocculation could not be assessed, but batch tests demonstrated the flocculation potential of this strain in the short term.