In situ detection of starch-hydrolyzing microorganisms in activated sludge

Polysaccharides constitute a significant part of the organic matter in domestic wastewater and their hydrolysis plays an important role in their transformation and nutrient removal in activated sludge wastewater treatment plants. However, there is no information available about the identity, ecophysiology, and abundance of starch-hydrolyzing organisms (SHOs) in these plants. In this study, fluorescence in situ enzyme staining with BODIPY fluorescein-labeled starch was applied and optimized to label SHOs expressing alpha-amylase in activated sludge plants. Fluorescence on the surface of bacteria expressing alpha-amylase activity was clearly visualized. In 11 full-scale nutrient-removing wastewater treatment plants examined, the morphotypes of the dominant SHOs were always cocci in clusters of tetrads, short rods in clusters, and some filamentous organisms. The SHOs were identified by combining in situ enzyme staining and FISH using a range of available oligonucleotide probes. All the SHOs observed were Actinobacteria, and most had the phenotype of polyphosphate-accumulating organisms closely related to the genus Tetrasphaera in the family Intrasporangiaceae. The SHOs were present in most of the wastewater treatment plants examined and comprised, in total, up to 11% of bacterial biovolume and thus formed an important part of the microbial communities.