The Microbial Database for Activated Sludge (MiDAS) field guide is a freely available online resource linking the identity of abundant and process critical microorganisms in activated sludge wastewater treatment systems to available data related to their functional importance. Phenotypic properties of some of these genera are described, but most are known only from sequence data. The MiDAS taxonomy is a manual curation of the SILVA taxonomy that proposes a name for all genus-level taxa observed to be abundant by large-scale 16 S rRNA gene amplicon sequencing of full-scale activated sludge communities. The taxonomy can be used to classify unknown sequences, and the online MiDAS field guide links the identity to the available information about their morphology, diversity, physiology and distribution. The use of a common taxonomy across the field will provide a solid foundation for the study of microbial ecology of the activated sludge process and related treatment processes. The online MiDAS field guide is a collaborative workspace intended to facilitate a better understanding of the ecology of activated sludge and related treatment processes—knowledge that will be an invaluable resource for the optimal design and operation of these systems.Database URL: http://www.midasfieldguide.org

Wastewater is increasingly viewed as a resource, with anaerobic digester technology being routinely implemented for biogas production. Characterising the microbial communities involved in wastewater treatment facilities and their anaerobic digesters is considered key to their optimal design and operation. Amplicon sequencing of the 16S rRNA gene allows high-throughput monitoring of these systems. The MiDAS field guide is a public resource providing amplicon sequencing protocols and an ecosystem-specific taxonomic database optimized for use with wastewater treatment facility samples. The curated taxonomy endeavours to provide a genus-level-classification for abundant phylotypes and the online field guide links this identity to published information regarding their ecology, function and distribution. This article describes the expansion of the database resources to cover the organisms of the anaerobic digester systems fed primary sludge and surplus activated sludge. The updated database includes descriptions of the abundant genus-level-taxa in influent wastewater, activated sludge and anaerobic digesters. Abundance information is also included to allow assessment of the role of emigration in the ecology of each phylotype. MiDAS is intended as a collaborative resource for the progression of research into the ecology of wastewater treatment, by providing a public repository for knowledge that is accessible to all interested in these biotechnologically important systems. Database URL: http://www.midasfieldguide.org

Filamentous bacteria belonging to the phylum Chloroflexi have received considerable attention in wastewater treatment systems for their suggested role in operational problem of impaired sludge settleability known as bulking. Their consistently high abundance in full-scale systems, even in the absence of bulking, indicates that they make a substantial contribution to the nutrient transformations during wastewater treatment. In this study, extensive 16S rRNA amplicon surveys of full-scale Danish WWTPs were screened to identify the most numerically important Chloroflexi genera. Fluorescence in situ hybridization probes were designed for their in situ characterization. All abundant phylotypes of the phylum were identified as facultative anaerobic chemoorganotrophs involved in fermentation of sugars. These groups were all filamentous but differed in their morphology and spatial arrangement. 'Candidatus Villigracilis' was predominantly located within the activated sludge flocs, where they possibly have structural importance, and their abundance was relatively stable. Conversely, the abundance of 'Candidatus Amarolinea' was highly dynamic, relative to other genera, sometimes reaching abundances in excess of 30% of the biovolume, suggesting their likely role in bulking episodes. This study gives an important insight into the role of Chloroflexi in WWTPs, thus contributing to the broader goal of understanding the ecology of these biotechnologically important systems.

Enhanced biological phosphorus removal (EBPR) involves the cycling of biomass through carbon-rich (feast) and carbon-deficient (famine) conditions, promoting the activity of polyphosphate accumulating organisms (PAOs). However, several alternate metabolic strategies, without polyphosphate storage, are possessed by other organisms, which can compete with the PAO for carbon at the potential expense of EBPR efficiency. The most studied are the glycogen accumulating organisms (GAOs), which utilize aerobically stored glycogen to energize anaerobic substrate uptake and storage. In full-scale systems the Micropruina spp. are among the most abundant of the proposed GAO, yet little is known about their ecophysiology. In the current study, genomic and metabolomic studies were performed on Micropruina glycogenica str. Lg2T and compared to the in situ physiology of members of the genus in EBPR plants using state-of-the-art single cell techniques. The Micropruina spp. were observed to take up carbon, including sugars and amino acids, under anaerobic conditions, which were partly fermented to lactic acid, acetate, propionate, and ethanol, and partly stored as glycogen for potential aerobic use. Fermentation was not directly demonstrated for the abundant members of the genus in situ, but was strongly supported by the confirmation of anaerobic uptake of carbon and glycogen storage in the absence of detectable polyhydroxyalkanoates or polyphosphate reserves. This physiology is markedly different from the classical GAO model. The amount of carbon stored by fermentative organisms has potentially important implications for phosphorus removal – as they compete for substrates with the Tetrasphaera PAO and stored carbon is not made available to the “Candidatus Accumulibacter” PAO under anaerobic conditions. This study shows that the current models of the competition between PAO and GAO are too simplistic and may need to be revised to take into account the impact of potential carbon storage by fermentative organisms.

18Filamentous bacteria belonging to the phylum Chloroflexi have received considerable 19 attention in wastewater treatment systems for their suggested role in operational 20 problem of impaired sludge settleability known as bulking. Their consistently high 21 abundance in full-scale systems, even in the absence of bulking, indicates that they 22 make a substantial contribution to the nutrient transformations during wastewater 23 treatment. In this study, extensive 16S rRNA amplicon surveys of full-scale Danish 24WWTPs were screened to identify the most numerically important Chloroflexi 25 genera. Fluorescence in situ hybridization probes were designed for their in situ 26 characterization. All abundant phylotypes of the phylum were identified as facultative 27 anaerobic chemoorganotrophs involved in fermentation of sugars. These groups were 28 all filamentous but differed in their morphology and spatial arrangement. 'Candidatus 29Villogracilis' was predominantly located within the activated sludge flocs, where they 30 possibly have structural importance, and their abundance was relatively stable. 31Conversely, the abundance of 'Candidatus Amarilinum' was highly dynamic, relative 32 to other genera, sometimes reaching abundances in excess of 30% of the biovolume, 33suggesting their likely role in bulking episodes. This study gives an important insight 34 into the role of Chloroflexi in WWTPs, thus contributing to the broader goal of 35 understanding the ecology of these biotechnologically important systems. 36