Abstract
Candidatus Microthrix is one of the most common bulking filamentous microorganisms found in activated sludge wastewater treatment plants (WWTPs) across the globe. One species, Ca. M. parvicella, is frequently observed, but global genus diversity, as well as important aspects of its ecology and physiology, are still unknown. Here, we use the MiDAS ecosystem-specific 16S rRNA gene database in combination with amplicon sequencing of Danish and global WWTPs to investigate Ca. Microthrix spp. diversity, distribution, and factors affecting their global presence. Only two species were abundant across the world confirming low diversity of the genus: the dominant Ca. M. parvicella and an unknown species typically present along with Ca. M. parvicella, although usually in lower abundances. Both species were mostly found in Europe at low-to-moderate temperatures and their growth was favored in municipal WWTPs with advanced process designs. As no isolate is available for the novel species, we propose the name “Candidatus Microthrix subdominans.” Ten high-quality metagenome-assembled genomes recovered from Danish WWTPs, including 6 representing the novel Ca. M. subdominans, demonstrated high genetic similarity between the two species with a likely preference for lipids, a putative capability to reduce nitrate and nitrite, and the potential to store lipids and poly-P. Ca. M. subdominans had a potentially more versatile metabolism including additional sugar transporters, higher oxygen tolerance, and the potential to use carbon monoxide as energy source. Newly designed fluorescence in situ hybridization probes revealed similar filamentous morphology for both species. Raman microspectroscopy was used to quantify the in situ levels of intracellular poly-P. Despite the observed similarities in their physiology (both by genomes and in situ), the two species showed different seasonal dynamics in Danish WWTPs through a 13-years survey, possibly indicating occupation of slightly different niches. The genomic information provides the basis for future research into in situ gene expression and regulation, while the new FISH probes provide a useful tool for further characterization in situ. This study is an important step toward understanding the ecology of Ca. Microthrix in WWTPs, which may eventually lead to optimization of control strategies for its growth in this ecosystem.
Highlights
The activated sludge process is the most common wastewater treatment technology applied worldwide
Using 16S rRNA gene amplicon sequencing data we investigated factors correlating to their global presence, and their influence on sludge settling properties
M. parvicella was dominant in the majority of the Danish and global plants, with the average read abundance of 1.7 ± 2.0% and 0.6 ± 1.2%, respectively
Summary
The activated sludge process is the most common wastewater treatment technology applied worldwide. M. parvicella are published: Dutch isolate Bio-17 (Muller et al, 2012), and Italian isolate RN1 (McIlroy et al, 2013) These genomes confirmed the physiology observed by in situ studies, showing assimilation and storage of LCFAs, the ability to store poly-P, and the potential to reduce nitrate and nitrite. Recent studies have granted valuable access to a high number of metagenome-assembled genomes (MAGs) representing microorganisms present in situ in the WWTP environment (Parks et al, 2017; Singleton et al, 2021) These MAGs together with high-throughput amplicon sequencing techniques provide a valuable tool set to explore the diversity of microbes present in the WWTP ecosystem. Our results provide important new information about this troublesome filamentous genus in the activated sludge ecosystem, which may help to develop more targeted strategies for its control
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