Phage treatment of filamentous bacteria in activated sludge

Abstract

The activated sludge process is one of the most commonly used wastewater treatment processes, which consists of a biochemical stage (aeration basin) and physical separation (secondary clarifier). The successful operation of this process relies on effective separation of the biomass from the treated wastewater. Under certain operating conditions, however, excessive growth of filamentous bacteria can cause sludge bulking and foaming, which limits solid-liquid separation, negatively impacting process performance. Currently, no effective treatment method exists that selectively controls the abundance of troublesome filamentous bacteria without adversely impacting the microbial ecology. The application of phages, viruses that infect and lyse bacteria, is potentially a target-specific and sustainable biocontrol method that could combat operational problems associated with the overabundance of filamentous bacteria; a strategy that has received increased interest over the past decade. Successful phage application, however, requires a better understanding of the influence of filamentous bacteria on the physical characteristics of the activated sludge flocs that impact solid-liquid separation and floc stability. Additionally, a better understanding of the adsorption of phages onto flocs and diffusion of phage within these structures is required, as these two factors will impact the efficacy and dynamics of phage treatment in the activated sludge process. The main objective of this research was therefore to investigate these factors and assess their influence on the application of phages in order to further develop phage as a potential treatment method for problematic filamentous bacteria associated with both foaming and sludge bulking.

In this project, a semi-automated image analysis method was developed and applied to quantify the abundance of filamentous bacteria in activated sludge. The length of protruding filaments was normalised to the floc area, which gives a relative measure of filament abundance. It was shown that a higher abundance of filaments enhanced the resistance of activated sludge flocs to shear-induced breakup, which is in agreement with the filamentous backbone theory. Flocs with a higher abundance of filamentous bacteria, however, also had an adverse impact on solid-liquid separation, increasing the resistance to settling, as quantified by the hindered settling function at solid concentrations below the gel point. An increased abundance of filaments also caused poorer sludge compactability, as reflected by lower gel point concentrations. From these observations it is evident that a balance between the filamentous and floc-forming bacteria is desired. Ideally, the abundance of filamentous bacteria should be sufficiently high to enhance floc stability but sufficiently low to prevent poor performance of solid-liquid separation processes. Importantly, this work refocuses the attention on the vital role of filamentous bacteria in floc stability, which is in agreement with the filamentous backbone theory.

The adsorption of phages to activated sludge flocs is an important consideration for phage treatment of filamentous bacteria that affects both the likelihood of individual infection and the dynamics of phage infection in the system but has not been studied to date. Systematic adsorption experiments were performed under controlled conditions using model alginate surfaces that resemble the extracellular polymeric substances (EPS) of activated sludge flocs. Theoretical interaction energies were calculated and compared to experimental deposition data to determine the dominant interactions between phage particles and the alginate surface. These calculations sought to examine both electrostatic and hydrophobic interactions. An increase in the concentration of counter ions resulted in a higher degree of electrostatic screening and enhanced phage deposition. The relatively high hydrophobicity of phage GTE6 also resulted in a significant contribution of hydrophobic attraction to the overall interaction energy. The attractive hydrophobic interaction counters the repulsive electrostatic interaction and results in more favourable conditions for phage adsorption. The adsorption of phage GTE6 to activated sludge flocs was also investigated by means of batch adsorption experiments. Kinetic and isotherm parameters of adsorption were determined, providing valuable insights regarding the application of phages to the activated sludge process. These parameters should be incorporated into dynamic models of phage treatment in the activated sludge process to further evaluate the impact of adsorption on the dynamics and efficacy of phage treatment for filamentous bacteria. It is important to take these effects into account when dosing strategies are developed for application at plant level.

The diffusion of phages into activated sludge flocs will also influence the effectiveness of phage treatment. Model systems consisting of alginate beads with immobilized Gordonia terrae were developed to demonstrate the influence of the matrix structure and the potential protective effect provided by the EPS of activated sludge flocs against phage infection. Alginate beads prepared by internal gelation were used as a model system to represent activated sludge flocs with a loose EPS matrix, whereas external gelation was used to represent flocs with a relatively dense EPS matrix or compact EPS layer surrounding the floc. The spread of infection and subsequent lysis of embedded host bacteria were measured by means of confocal scanning laser microscopy and fluorescent viability staining. The matrix structure of alginate beads prepared by internal gelation permitted diffusion of phage GTE6 and subsequent infection of embedded host bacteria. The presence of the alginate matrix resulted in slower diffusion of phages and subsequently slower infection kinetics compared to dispersed bacteria in the absence of a matrix structure. A denser matrix at the periphery of alginate beads, prepared by external gelation, prevented diffusion of phage GTE6 into the alginate beads and therefore protected the embedded host bacteria against phage infection. The results from these different systems, which potentially represent activated sludge flocs with low and high solids retention times, suggest that phage diffusion into flocs may be restricted by the denser EPS matrix of flocs associated with longer retention times. Under such conditions, the filamentous backbone within the confines of the flocs will be protected against phage infection, while free-floating and protruding filamentous bacteria, which are responsible for foaming or bulking when abundant, are readily accessible to phages for infection and subsequent lysis. This finding impacts the dynamics of phage infection and should be considered in model development and future implementation plans.

Overall, the knowledge gained through this thesis provides a better understanding of practical aspects that will impact the application of phages to control filamentous bacteria in the activated sludge process. This research confirms that filamentous bacteria are necessary to enhance floc stability but if excessively abundant will adversely affect solid-liquid separation. Furthermore, this research has improved our understanding of phage adsorption to EPS and activated sludge flocs, factors that will impact the dynamics and effectiveness of phage treatment in the activated sludge process. The model systems applied in this work illustrated the impact of the EPS matrix structure on the accessibility of filamentous bacteria within the confines of activated sludge flocs, which will have a significant influence on the efficacy of phage treatment and impact process performance. This improved understanding will assist modelling of the system and guide the development of phage dosing strategies to reduce the risks associated with the implementation of phage treatment at plant level. The toolsets and framework provided in this thesis should be applied in further application studies. The semi-automated image analysis method can be used for routine quantitative assessment of filament abundance at plant level, which reduces operator bias and is useful for improved process monitoring and research purposes. Additionally, a useful framework was developed for phage application studies in which the 3-dimensional growth of bacteria and spread of phage infection in alginate matrices can be investigated by means of confocal laser scanning microscopy. This technique will be valuable in the broader field of phage biology, for example in the application of phage to other flocculated or biofilm systems.