Identifying mechanisms for enhanced bacterial granulation using pure culture isolates and bench scale batch reactor systems.

Treatment of wastewater is imperative for avoiding the spread of disease and preventing environmental imbalances. Traditionally, activated sludge is used to treat a variety of wastewaters, however, microbial granules offer more efficient treatment and have been widely studied in the context of engineering principals and reactor design. This thesis presents insights into granulation by applying molecular and microbial ecology approaches to the process of granule formation. Differential growth morphologies of strains with identical 16S rRNA gene sequences were used to investigate the molecular mechanisms affecting aggregation. Conditions that promoted aggregation were explored, with calcium ion addition significantly increasing aggregation of one isolate in particular, NS011. Exposure of isolate NS011 to a range of enzymes revealed proteins and polysaccharides to be responsible for maintaining aggregate stability. Four mutants with altered aggregation phenotypes were identified from a Tn5 transposon library constructed from isolate NS011. The sequencing of interrupted genes revealed homology to the extracellular protein, GumC, and putative extracellular proteins of Xanthomonas species, confirming the role of extracellular proteins in intercellular interactions. Further, a simplified batch reactor system was developed to assess the roles of extracellular DNA (eDNA), protozoan predation and intercellular signalling in granulation. Aggregate size determination was performed in conjunction with genetic fingerprinting and pyrosequencing for bacterial community composition analysis. While eDNA addition did not affect granulation, treatment with DNase significantly reduced granule size, suggesting a role for eDNA in maintenance of granules. Similarly, results from predation experiments suggested that the removal of grazing pressure by the ciliate, Tetrahymena pyriformis, significantly decreased aggregate size, while T. pyriformis addition altered the microbial community composition. The addition of a cocktail of acylated homoserine lactone signalling molecules had no significant effect on aggregation or community composition.The work described in this thesis highlights the importance of studying mechanisms of intercellular interactions in defined systems, to enhance our understanding, and thus control of the granulation process. This work also demonstrates the effect of environmental factors on sludge community composition, indicating the importance of such factors in granule development, and offers the batch reactor system as a simplified method to further investigate the impact of ecological factors on granulation