From MAGs to riches: exploring the microbial community of activated sludge using 1,083 high-quality metagenome-assembled genomes

Caitlyn Singleton (Aalborg University, Denmark) opened the Microbiome breakout session by introducing her work studying activated sludge – the microbial process used worldwide to treat wastewater. Without this treatment process, she explained, pathogens would remain present and could pose a public health risk. Furthermore, nutrients such as phosphate and nitrogen would go on to cause problems such as eutrophication; instead, these nutrients can be recovered and used in agriculture. Activated sludge can be processed further via anaerobic digestion which, via anaerobic microorganisms, can produce biogas, heat and energy which can be fed back into the National Grid.

To analyse the complex communities of microorganisms undertaking these processes, Caitlyn and her team employ metagenomic sequencing, using the PromethION platform to recover high-quality genome sequences. Their aim was to recover over 1000 high-quality metagenome-assembled genomes (MAGs) from novel, uncultivated populations. This would enable functional analysis, to determine their metabolic potential and possible roles in the community. Furthermore, analysis of the full-length 16S regions would allow cross-referencing with their 13-year database of amplicon data, providing information on the abundance of microbes over time. This, in turn, would allow the design of FISH probes to explore their morphology and confirm their functions.

Designing their experiment, Caitlyn and her team selected 23 wastewater treatment plants (WWTPs) for nanopore sequencing, complementing this with DNA collected in 2016 and 2017 from the same WWTPs. Nanopore sequencing yielded 1.08 Tb of data; 0.92 Tb of short-read data was also generated for polishing. The earlier data sets comprised shallow-depth short-read sequencing. Using a bioinformatics pipeline developed by Rasmus Kirkegaard, the team recovered 3,733 medium- to high-quality MAGs and 1,045 true high-quality MAGs featuring full-length 16S regions. They also identified 38 reduced-genome bacteria and archaea, including one Paceibacteria genome: an exciting find, as organisms from this group had not previously been characterised in situ. Using the 16S sequences, they were able to design FISH probes and visualise the bacterium; assembly of the genome also identified versions with and without an additional fragment which was revealed to represent a phage.

Analysis of the >1k high-quality MAGs revealed 581 species across 30 bacterial and 2 archaeal phyla. They then selected an abundant lineage, from a novel genus, to investigate polyphosphate accumulation. Designing FISH probes from the 16S data, they visualised the bacteria in situ, revealing rod morphology and determining their abundance. Using microspectroscopy, they identified polyphosphate stored at high levels, similar to known polyphosphate-accumulating organisms. Further characterisation included identification of the organism as a probable methylotroph: they have suggested a name of Candidatus Methylophosphatis. Concluding her talk, Caitlyn highlighted how nanopore sequencing recovered ~30% of the metagenome communities in high-quality MAGs, identified a novel key genus, and enabled further in situ experiments.