In this study, Illumina sequencing was used for the identification of bulking and foaming bacteria in industrial wastewater treatment plants. The reliable identification of bulking and foaming bacteria represents the first step in developing effective and specific control strategies to avoid disturbances in activated sludge systems. Illumina sequencing revealed 432 16S rRNA operational taxonomic units, representing phylotypes and including 21 bulking and foaming bacteria in the two investigated industrial wastewater treatment plants. Foaming represents the most severe problem in the cascade biology system. Up to 22.5% of all sequencing reads are bulking and foaming bacteria, including Chryseobacterium, Candidatus Microthrix parvicella and Gordonia sp. as the dominant bulking and foaming bacteria which are known for foam formation. Moreover, Illumina sequencing revealed an increase in Candidatus Microthrix parvicella and Gordonia sp. reads from activated sludge to foam and scum samples, indicating a preferred flotation and/or growth advantages in the foam and scum layers. Analyses of the taxonomic assignment and distribution showed that the phylum Actinobacteria is the most dominant phylum, underlining the key role of Actinobacteria in bulking and foaming. Multivariate data analysis was applied, revealing that the dominant bulking and foaming bacteria are positively correlated with the sludge age and influent flow and negatively correlated with the dissolved oxygen level and the temperature. In terms of developing a specific control strategy, the positive linear relationships to the fatty acid and surfactant sludge loadings are highlighted and the removal of lipid compounds from the wastewater influent could avoid an overgrowth of bulking and foaming bacteria. Keywords CandidatusMicrothrix parvicella Á Filamentous bacteria Á Filamentous foaming Á Illumina sequencing Á Next-generation sequencing Á Industrial activated sludge plants Editorial responsibility: BV Thomas.

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The present study demonstrates the application of in situ microscopy for monitoring the growth of filamentous bacteria which can induce disturbances in an industrial activated sludge process. An in situ microscope (ISM) is immersed directly into samples of activated sludge with Microthrix parvicella as dominating species. Without needing further preparatory steps, the automatic evaluation of the ISM-images generates two signals: the number of individual filaments per image (ISM-filament counting) and the total extended filament length (TEFL) per image (ISM-online TEFL). In this first version of the image-processing algorithm, closely spaced crossing filament-segments or filaments within bulk material are not detected. The signals show highly linear correlation both with the standard filament index and the TEFL. Correlations were further substantiated by comparison with real-time polymerase chain reaction (real-time PCR) measurements of M. parvicella and of the diluted sludge volume index. In this case study, in situ microscopy proved to be a suitable tool for straightforward online-monitoring of filamentous bacteria in activated sludge systems. With future adaptation of the system to different filament morphologies, including cross-linking filaments, bundles, and attached growth, the system will be applicable to other wastewater treatment plants.

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