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Abstract
Chitin is one of the most abundant biopolymers on Earth. In this thesis, the colonisation of chitin by activated sludge bacteria was explored. Chitin was incubated with pure cultures of Aeromonas hydrophila GC1 isolated from activated sludge and with activated sludge itself. Stages of biofilm development were monitored by CLSM and SEM. Biomass accumulation was assessed by DNA yields. Denaturing Gradient Gel Electrophoresis and pyrosequencing were used to characterise bacterial communities colonising chitin. NSI-MS, TLC, and bioassays were used to detect AHL production on the surface of chitin. Aeromonas (pBB-luxR), a GFP based monitor strain, was also employed. Chitin degradation was monitored by SEM. Chitinase activity was detected using a colorimetric chitinase assay.
Results showed that cells attach to chitin after 24 hours of incubation. DNA yields revealed that biomass of A. hydrophila on chitin increases after 24 hours of incubation and decreases after 200 hours. Microscopy showed that integrity of the chitin becomes disrupted after 288 hours of being incubated in sludge. Results revealed that members of the chitinophagaceae family, of the bacteroidetes phylum, are the most abundant bacteria in sludge incubated with chitin. The monitor strain assay proved to be the most suitable method for AHL detection. AHLs were detected on chitin pieces at 24 hours, before cell attachment to chitin was observed. Chitinase activity was detected after 24 hours. These results supported the proposed model for chitin colonisation; where AHLs that bind and coat chitin are produced, followed by bacterial colonisation of the chitin surface and up-regulation of chitinase expression.