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The purpose of this dissertation was to assess the critical role of extracellular polymeric substances (EPS) in the photogranulation of activated sludge, in a hydrostatic environment. The first section evaluates the fate and dynamics of different fractions of EPS in sludge-based photogranulation under hydrostatic conditions. The study shows that during the transformation of activated sludge into a photogranular biomass, sludge’s base-extractable proteins selectively degrade. Strong correlations between base-extracted proteins and the growth of chlorophyll a and chlorophyll a/b ratio suggest that the bioavailability of this organic nitrogen is linked with selection and enrichment of filamentous cyanobacteria under hydrostatic conditions. The results of soluble and sonication-extractable EPS and microscopy also show that the growth of filamentous cyanobacteria required large amounts of polysaccharide-based EPS for their motility and maintenance. With findings on the progression of photogranulation, the fate and dynamics of EPS, and microscopy on microstructures associated with EPS, potential mechanisms of photogranulation occurring under hydrostatic conditions are discussed. The second section evaluates and shows that multiple EPS extraction methods are required in order to characterize EPS during the transformation of activated sludge into a photogranule in a hydrostatic environment. The present study reveals why cyanobacteria are selected and how different fractions of EPS and their recycle leads to photogranulation in hydrostatic conditions. Despite differences in sludge inoculum, EPS extraction using five different methods, centrifugation, cation exchange resin (CER), base, sonication, and heat, show that trends are significantly similar, statistically, between two sludge sources (Amherst and Hadley). The results presented above show that different EPS extraction methods are required to capture different fractions of EPS with respect to protein, polysaccharide, and humic acid composition and organic carbon and nitrogen content. EPS extraction methods for polysaccharides was found to be the most biased, followed by humic acids, then proteins. This suggests that different methods target different EPS fractions (more associated with polysaccharides), but may share overlap between the methods (proteins and humic acids). All methods had statistically significant moderate to strong correlations with one or more constituents, chlorophylls, nitrogen species, and select cations and anions, which have been previously established as strong indicators of successful granule formation. These results suggest that different EPS fractions are linked to multiple processes during hydrostatic photogranulation, including the enrichment of filamentous cyanobacteria, nitrogen metabolism and recycle of organic nitrogen, assimilation and biofilm incorporation of ammonium (NH4+-N), and biofilm structure, further suggesting that the role of EPS is a complex process with multiple courses of action. The final section focuses on the addition and role of cations for the enhancement of activated sludge photogranulation in a hydrostatic environment. This study observed that the addition of monovalent (sodium-Na+) and divalent cations (Calcium-Ca2+ and Magnesium- Mg2+), at specific concentrations 10-40 meq/L, leads to a higher percentage of total and spherical granules, in comparison to light control (no cation amendment) and dark control cultivations. Based on crude EPS, ammonium sulfate precipitation, and sulfate polyacrylamide gel electrophoresis (SDS-PAGE) results, Light+Ca2+ treatments show greater recovery of CER protein after ASP, and different recovery patterns in comparison to light and dark control, suggesting that more hydrophobic protein is available. This further infers that the addition of Ca2+ may influence the hydrophobicity of EPS proteins during hydrostatic photogranulation. After ASP, SDS-PAGE was applied and banding pattern across the treatments showed same the EPS protein on a molecular level which did not change with the addition of Ca2+ (in comparison to control). This may further imply that enhancement is more likely due to Ca2+ cation bridging, versus changes on a molecular level influenced by the microbial community.
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