Diversity and abundance of filamentous and non-filamentous "Leptothrix" in global wastewater treatment plants

Species belonging to the genus Leptothrix are widely distributed in the environment and in activated sludge (AS) wastewater treatment plants (WWTPs). They are commonly found in iron-rich environments and reported to cause filamentousbulking in WWTPs. In this study, the diversity, distribution, and metabolic potential of the most prevalent Leptothrix spp. found in AS worldwide were studied. Our 16S rRNA amplicon survey showed that Leptothrix belongs to the general core community of AS worldwide, comprising 32 species with four species being most commonly found. Their taxonomic classification was re-evaluated based on both 16S rRNA gene and genome-based phylogenetic analysis showing that three of the most abundant "Leptothrix" species represented species in three other genera, Rubrivivax, Ideonella, and the novel genus, Ca. Intricatilinea. New fluorescencein situ hybridization (FISH) probes revealed rod-shaped morphology for the novel Ca. Rubrivivax defluviihabitans and Ca. Ideonella esbjergensis, while filamentous morphology was found only for Ca. Intricatilinea gracilis. Analysis of high-quality metagenome-assembled genomes revealed metabolic potential for aerobic growth, fermentation, storage of intracellular polymers, partial denitrification,photosynthesis, and iron reduction. FISH in combination with Raman microspectroscopy confirmed the in situ presence of chlorophyll and carotenoids in Ca. Rubrivivax defluviihabitans and Ca. Intricatilinea gracilis. This study resolves the taxonomy of abundant but poorly classified "Leptothrix" species, providing important insights into their diversity, morphology, and function in global AS wastewater treatment systems