Enzymatic hydrolysis under different electron acceptor conditions in nutrient removal activated sludge treatment processes is a weak link in the Activated Sludge Model no. 2 (Henze et al., 1995). An experimental study was undertaken to gain insight into the hydrolysis process with specific focus on hydrolysis kinetics and rates under different electron acceptor conditions. Two pure cultures, Bacillus amyloliquefaciens (Gram positive) and Pseudomonas saccharophila (Gram negative) were chosen for the study. In addition, activated sludge grown in an anaerobic-aerobic system was tested for enzymatic activity using starch as the model substrate. The hydrolytic enzymes were found to be released into the bulk in pure cultures whereas the enzyme activity was found to be mainly associated with the cell surfaces in activated sludge. Further, it was observed that the development of the hydrolytic enzyme system in Bacillus amyloliquefaciens and P. saccharophila is strongly suppressed under anoxic and anaerobic conditions. However, the effect of anaerobic and aerobic incubation on hydrolytic enzyme activity in activated sludge was found to be small. Starch hydrolysis kinetic data from batch experiments with activated sludge followed substrate saturation kinetics that were linear with biomass concentration. Finally, the similar hydrolytic enzyme activities observed under anaerobic and aerobic phases of a sequencing batch reactor are explained by considering the aspects of enzyme location and enzyme system development under aerobic and anaerobic phases. It is proposed that the floc bound enzymes are recycled in a single sludge system so that an equilibrium exists between enzyme loss and synthesis at steady state.

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