Morphological and molecular identification of filamentous microorganisms associated with bulking and foaming activated sludge

Abstract

The activated sludge process comprises a complex and enriched culture of a mixture of generalist and specialist organisms. The lack of knowledge on species diversity of microbial communities is due to the simplicity of bacterial morphology and the phenotypic characters, and the unculturable portion of microbial cells in natural habitats. Although a wide range of bacteria can be isolated using conventional microbiological techniques of sample dilution and spread plate inoculation, many well-known activated sludge bacteria can not be isolated using them. The individual microbial cells in activated sludge grow in aggregates that consist of floc-forming organisms together with filamentous microorganisms that form the backbone of the activated sludge floes. Overgrowth of these filamentous microorganisms often causes settling problems called bulking and foaming. These problems consist of slow settling, poor compaction of solids and foam overflow into the effluent. Although methods for the isolation of filamentous bacteria from mixed liquor samples have been investigated, the attempts have been largely unsuccessful. In this study we investigated bulking and foaming activated sludge to identify the dominant filamentous organisms using microscopy and molecular techniques. Using microscopy, the dominant filament associated with the foaming sample was "Microthrix parvicella" and in the bulking sample was Nocardia spp. The foaming sample was investigated using molecular techniques that involved 165 rDNA sequencing. Although some of the clones isolated from the sludge foam were associated with filamentous bacteria causing foam, no positive identification could be made. In the part of the study that was conducted in Australia, a rRNA-targeted oligonucleotide probe was designed for the identification of a filamentous organism occurring in activated sludge foam. This organism resembled Eikelboom Type 0041 and was classified in the candidate bacterial division TM7. The discrepancy that the sequence data did not indicate the dominant filamentous organisms observed by microscopy, highlights the fact that natural microbial communities need to be studied using a combination of techniques since none of the techniques available are sufficient to determine the complete community structure of complex communities such as activated sludge.