Abstract
Enhanced biological phosphorus removal (EBPR) is an efficient and cost-effective technology for wastewater treatment, with the potential to be coupled with resource recovery. The main challenge hindering a wider implementation of EBPR is that the process can experience performance variability or upsets, often affecting the microbial consortium underpinning wastewater treatment, and compromising effluent quality. Phages, viruses that predate on bacteria, have been demonstrated to affect treatment performance by infection of key functional bacteria in the system. Withal, knowledge of phage ecology of EBPR, or biological wastewater treatment (BWT) in general, is very limited and many questions are still unanswered about the role of phages in engineered microbial communities. This thesis aims to quantify viruses in BWT systems, identify and characterise phages specific to key EBPR organisms, and propose new methods for their propagation without the need for pure cultures.The literature review on the occurrence, characterization, and function of phages in BWT highlighted the challenges necessary to be overcome. Two of the main gaps identified - namely the meager information regarding viruses abundance and dynamics along BWT systems and the lack of an established method for the recovery and quantification of viruses – were addressed in Chapter 3. The quantification of viruses at different treatment stages of five full-scale BWT systems was performed using flow cytometry, with SYBR Green I staining and including an elution step. The results showed a range of concentration of viral-likeparticles (VLP) from 1.47 x 107 to 2.25 x 1010 VLP mL-1, with the highest concentrations in the influent and then a slight decrease in the concentration of viruses along the treatment process. This chapter is one of the few studies investigating the concentration of viruses in different units of the treatment process.
In addition, a similar quantification was done for Decholoromas specific phages using plaque-assay (Chapter 3). Bacteria belonging to the Dechloromonas genus are found in high relative abundances in fullscale EBPR systems and present a phenotype compatible with nutrient removal from wastewater. Phages infecting Dechloromonas were found in the influent and in the anaerobic, anoxic and aerobic tanks of secondary treatment, with a decrease observed along the treatment process. Isolation experiments resulted in a total of 12 phages isolates from samples collected in BWT plants. The phages were further characterized by the description of the plaques of infection and morphology observed with transmission electron microscopy. A total of 9 out of the 12 isolates were used in infection batch tests with the host to assess the dynamics of infection. The work described in Chapter 4 is the first report on the isolation of phages infecting Dechloromonas, and the same culture-dependent strategy can be applied using hosts that are related to key bacteria in EBPR if available as pure culture.
The use of culture-dependent methods still limits the study of specific phages or bacteria, and novel strategies are required to assess unculturable organisms. Since phages thrive and propagate in the presence of the respective hosts, even if within a microbial consortium, we hypothesized that microbial communities enriched in a particular group of bacteria could favour the propagation of specific phages. Chapter 5 describes the preliminary work done on developing this strategy. A lab-acclimatized EBPR mixed microbial culture was used to support the propagation of phages within various viruses suspensions recovered from BWT samples. The successful propagation of phages was further demonstrated by infection of phage-free, enriched microbial cultures.
The concluding remarks highlight that there is still scope to apply established and widely used techniques for phage identification and isolation in the context of BWT. This can be an important contribution to foundational knowledge on phage ecology of wastewater treatment. Furthermore, it explains that the novel approaches demonstrated in this thesis have the potential to be applied to other organisms and wastewater treatment systems. Such approaches can be leveraged as tools to study the microbial ecology of EBPR and contribute to the improvement and wider implementation of this technology.
| Date of Award | 29 Mar 2023 |
|---|---|
| Original language | English |
| Awarding Institution |
|
| Supervisor | Ana Lanham (Supervisor), Brian Jones (Supervisor), Jannis Wenk (Supervisor) & Simon Bengtsson (Supervisor) |