The Microbial Ecology of Granular Sludge in Enhanced Biological Phosphorus Removal

Jeremy Barr (2010). The Microbial Ecology of Granular Sludge in Enhanced Biological Phosphorus Removal PhD Thesis, School of Chemical Engineering, The University of Queensland.

       
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Author Jeremy Barr
Thesis Title The Microbial Ecology of Granular Sludge in Enhanced Biological Phosphorus Removal
School, Centre or Institute School of Chemical Engineering
Institution The University of Queensland
Publication date 2010-11
Thesis type PhD Thesis
Supervisor Dr. Phillip Bond
Dr. Gene Tyson
Total pages 186
Total colour pages 24
Total black and white pages 162
Subjects 03 Chemical Sciences
Abstract/Summary The removal of unwanted nutrients, such as carbon, nitrogen and phosphorus from wastewater, is crucial in maintaining our waterways and preventing eutrophication. The enhanced biological phosphorus removal (EBPR) process is a widely applied and globally important biological process for the removal of phosphorus and treatment of wastewater. This removal performance occurs through the intracellular accumulation of phosphate by polyphosphate-accumulating organisms (PAO). Candidatus “Accumulibacter phosphatis” (Accumulibacter) are identified as a major PAO responsible for EBPR by culture-independent methods. Maintenance of stable EBPR performance is of importance and EBPR failure is often attributed to the presence of glycogen-accumulating organisms (GAO). Candidatus “Competibacter phosphatis” (Competibacter) has been identified as a major GAO, which competes anaerobically with PAO for carbon without removing phosphate, therefore negatively impacting EBPR performance. Typically, full-scale EBPR systems operate as floccular activated sludge, in which small biofilm aggregates (30-200 mm) of microorganisms treat wastewater. However, there is recent interest to operate activated sludge as ‘aerobic’ granules, which form larger biofilm aggregates (200-2000 mm) which exhibit faster settling characteristics and higher biomass concentrations than floccular sludge, making it operationally and economically advantageous. Phylogenetic-group specific probes for environmental samples. Since EBPR systems are mixed microbial communities, most studies are limited to in-situ investigations. The first research paper (Chapter 4) assessed the microbial community using fluorescence in-situ hybridisation (FISH). FISH is regularly used to assess the microorganisms present in EBPR activated sludge biomass, and to link process performance with resident microbial communities. This assessment has substantial implications for EBPR, specifically relating to the identification of two critical, relatively-abundant and competing bacterial populations of Accumulibacter and Competibacter. These are members of the Betaproteobacteria and Gammaproteobacteria respectively and targeted by the phylogenetic group specific (PGS) oligonucleotide FISH probes, BET42a and GAM42a. A metagenomic study of two EBPR biomasses, which produced a near complete genome for Accumulibacter, and our own 16S-23S rRNA gene analysis revealed that Accumulibacter was actually targeted by the Gammaproteobacteria PGS FISH probe. This FISH probe inconsistency was of particular importance on this occasion as the trusting use of the PGS probes would confuse the monitoring of key bacteria that have competing roles within EBPR. Granule formation mechanisms. To date, the majority of research on granular sludge has focused on optimisation of engineering aspects relating to reactor operation with little emphasis on the fundamental microbiology. The second research paper (Chapter 5) investigated microbial mechanisms for granule formation as observed in three laboratory-scale sequencing batch reactors operating for biological phosphorus removal, treating two different types of wastewater. During the initial stages of granulation two distinct granule types (white and yellow) were distinguished within the mixed microbial population. White granules appeared as compact, smooth, dense aggregates dominated by 97.5% Accumulibacter and yellow granules appeared as loose, rough, irregular aggregates with a mixed microbial population of 12.3% Accumulibacter and 57.9% Competibacter among other bacteria. Phylogenetic and microscopic analysis suggests the granule types are likely not a result of strain-associated differences, and rather the result of two different formation mechanisms. Further understanding and application of these formation mechanisms and the associated microbial ecology may provide conceptual information benefiting start-up procedures for full-scale granular sludge reactors. Bacteriophage activity causing community and performance changes. Bacteria are known to play important roles in biogeochemical cycles and biotechnology processes, such as EBPR, but little is known about the influence of bacteriophage on these processes. The third research paper (Chapter 6) investigated the effect of bacteriophage infection of EBPR process performance and granular sludge. However, a major impediment to the study of host-bacteriophage interactions is that the host bacteria and their bacteriophage are often not available in pure culture. In this study we used in-situ microbial methods to investigate a bacteriophage infection of the uncultured bacterium Accumulibacter, resulting in a decline in phosphorus removal performance and granular sludge stability within laboratory-scale EBPR treatment reactor. This is the first demonstration of bacteriophage affecting both bacterial-community structure and nutrient-removal performance in activated sludge and suggests that bacteriophage play a significant role in determining the structure and function of bacterial communities in activated sludges. Metatranscriptomic and metaproteomic comparison of floccular and granular sludge. Our understanding of the fundamental microbial mechanisms associated with granular sludge formation and stability is still very limited. The fourth research paper (Chapter 7) we present a combined metatranscriptomic and metaproteomic comparison between floccular and granular sludge and identifies significant differences in gene expression and protein. Overall, 569 mRNA transcripts and 191 proteins were identified as being statistically different between floccular and granular sludge. Results indicate that genes and proteins involved in signal transduction, cell motility, EPS formation and cell membrane biogenesis may play crucial roles in granulation. The study also identified a large number of hypothetical and unknown proteins, which may be involved in granule formation and stability, further emphasising our lack of understanding of granulation.
Keyword Accumulibacter
Bacteriophage
Enhanced Biological Phosphorus Removal (ebpr)
Floccular sludge
Granular activated sludge
Wastewater
Additional Notes Colour Pages; 21, 27, 30, 32, 48, 59, 73, 74, 77, 79, 85, 94, 102, 125, 167-176 Landscape Pages; 53, 73, 74, 79, 126, 177-185

 
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