Diversity and Ecophysiology of the Genus OLB8 and Other Abundant Uncultured Saprospiraceae Genera in Global Wastewater Treatment Systems

The Saprospiraceae family within the phylum Bacteroidota is commonly present and highly abundant in wastewater treatment plants (WWTPs) worldwide, but little is known about its role. In this study, we used MiDAS 4 global survey with samples from 30 countries to analyze the abundance and distribution of members of Saprospiraceae. Phylogenomics were used to delineate five new genera from a set of 31 high-quality metagenome-assembled genomes from Danish WWTPs. Newly designed probes for fluorescence in situ hybridization (FISH) revealed rod-shaped morphologies for all genera analyzed, including OLB8, present mostly inside the activated sludge flocs. The genomes revealed potential metabolic capabilities for the degradation of polysaccharides, proteins, and other complex molecules; partial denitrification; and storage of intracellular polymers (glycogen, polyphosphate, and polyhydroxyalkanoates). FISH in combination with Raman microspectroscopy confirmed the presence of intracellular glycogen in Candidatus Brachybacter, Candidatus Parvibacillus calidus (both from the former genus OLB8), and Candidatus Opimibacter, and the presence of polyhydroxyalkanoates in Candidatus Defluviibacterium haderslevense and Candidatus Vicinibacter. These results provide the first overview of the most abundant novel Saprospiraceae genera present in WWTPs across the world and their potential involvement in nutrient removal and the degradation of macromolecules

Reevaluation of the Phylogenetic Diversity and Global Distribution of the Genus "Candidatus Accumulibacter"

“Candidatus Accumulibacter” was the first microorganism identified as a polyphosphate-accumulating organism (PAO) important for phosphorus removal from wastewater. Members of this genus are diverse, and the current phylogeny and taxonomic framework appear complicated, with most publicly available genomes classified as “Candidatus Accumulibacter phosphatis,” despite notable phylogenetic divergence. The ppk1 marker gene allows for a finer-scale differentiation into different “types” and “clades”; nevertheless, taxonomic assignments remain inconsistent across studies. Therefore, a comprehensive reevaluation is needed to establish a common understanding of this genus, in terms of both naming and basic conserved physiological traits. Here, we provide this reassessment using a comparison of genome, ppk1, and 16S rRNA gene-based approaches from comprehensive data sets. We identified 15 novel species, along with “Candidatus Accumulibacter phosphatis,” “Candidatus Accumulibacter delftensis,” and “Candidatus Accumulibacter aalborgensis.” To compare the species in situ, we designed new species-specific fluorescence in situ hybridization (FISH) probes and revealed their morphology and arrangement in activated sludge. Based on the MiDAS global survey, “Ca. Accumulibacter” species were widespread in wastewater treatment plants (WWTPs) with phosphorus removal, indicating process design as a major driver for their abundance. Genome mining for PAO-related pathways and FISH-Raman microspectroscopy confirmed the potential for PAO metabolism in all “Ca. Accumulibacter” species, with detection in situ of the typical PAO storage polymers. Genome annotation further revealed differences in the nitrate/nitrite reduction pathways. This provides insights into the niche differentiation of these lineages, potentially explaining their coexistence in the same ecosystem while contributing to overall phosphorus and nitrogen removal. IMPORTANCE “Candidatus Accumulibacter” is the most studied PAO, with a primary role in biological nutrient removal. However, the species-level taxonomy of this lineage is convoluted due to the use of different phylogenetic markers or genome sequencing approaches. Here, we redefined the phylogeny of these organisms, proposing a comprehensive approach which could be used to address the classification of other diverse and uncultivated lineages. Using genome-resolved phylogeny, compared to phylogeny based on the 16S rRNA gene and other phylogenetic markers, we obtained a higher-resolution taxonomy and established a common understanding of this genus. Furthermore, genome mining of genes and pathways of interest, validated in situ by application of a new set of FISH probes and Raman microspectroscopy, provided additional high-resolution metabolic insights into these organisms

Re-evaluation of the phylogenetic diversity and global distribution of the genus Candidatus Accumulibacter

“Candidatus Accumulibacter” was the first microorganism identified as a polyphosphate-accumulating organism (PAO) important for phosphorus removal from wastewater. Members of this genus are diverse, and the current phylogeny and taxonomic framework appear complicated, with most publicly available genomes classified as “Candidatus Accumulibacter phosphatis,” despite notable phylogenetic divergence. The ppk1 marker gene allows for a finer-scale differentiation into different “types” and “clades”; nevertheless, taxonomic assignments remain inconsistent across studies. Therefore, a comprehensive reevaluation is needed to establish a common understanding of this genus, in terms of both naming and basic conserved physiological traits. Here, we provide this reassessment using a comparison of genome, ppk1, and 16S rRNA gene-based approaches from comprehensive data sets. We identified 15 novel species, along with “Candidatus Accumulibacter phosphatis,” “Candidatus Accumulibacter delftensis,” and “Candidatus Accumulibacter aalborgensis.” To compare the species in situ, we designed new species-specific fluorescence in situ hybridization (FISH) probes and revealed their morphology and arrangement in activated sludge. Based on the MiDAS global survey, “Ca. Accumulibacter” species were widespread in wastewater treatment plants (WWTPs) with phosphorus removal, indicating process design as a major driver for their abundance. Genome mining for PAO-related pathways and FISH-Raman microspectroscopy confirmed the potential for PAO metabolism in all “Ca. Accumulibacter” species, with detection in situ of the typical PAO storage polymers. Genome annotation further revealed differences in the nitrate/nitrite reduction pathways. This provides insights into the niche differentiation of these lineages, potentially explaining their coexistence in the same ecosystem while contributing to overall phosphorus and nitrogen removal. IMPORTANCE “Candidatus Accumulibacter” is the most studied PAO, with a primary role in biological nutrient removal. However, the species-level taxonomy of this lineage is convoluted due to the use of different phylogenetic markers or genome sequencing approaches. Here, we redefined the phylogeny of these organisms, proposing a comprehensive approach which could be used to address the classification of other diverse and uncultivated lineages. Using genome-resolved phylogeny, compared to phylogeny based on the 16S rRNA gene and other phylogenetic markers, we obtained a higher-resolution taxonomy and established a common understanding of this genus. Furthermore, genome mining of genes and pathways of interest, validated in situ by application of a new set of FISH probes and Raman microspectroscopy, provided additional high-resolution metabolic insights into these organisms

Low Global Diversity of Candidatus Microthrix, a Troublesome Filamentous Organism in Full-Scale WWTPs

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

Identification of microorganisms responsible for foam formation in mesophilic anaerobic digesters treating surplus activated sludge

Foaming is a common operational problem in anaerobic digestion (AD) systems, where hydrophobic filamentous microorganisms are usually considered to be the major cause. However, little is known about the identity of foam-stabilising microorganisms in AD systems, and control measures are lacking. This study identified putative foam forming microorganisms in 13 full-scale mesophilic digesters located at 11 wastewater treatment plants in Denmark, using 16S rRNA gene amplicon sequencing with species-level resolution and fluorescence in situ hybridization (FISH) for visualization. A foaming potential aeration test was applied to classify the digester sludges according to their foaming propensity. A high foaming potential for sludges was linked to the abundance of species from the genus Candidatus Microthrix, immigrating with the feed stream (surplus activated sludge), but also to several novel phylotypes potentially growing in the digester. These species were classified to the genera Ca. Brevefilum (Ca. B. fermentans) and Tetrasphaera (midas_s_5), the families ST-12K33 (midas_s_22), and Rikenellaceae (midas_s_141), and the archaeal genus Methanospirillum (midas_s_2576). Application of FISH showed that these potential foam-forming organisms all had a filamentous morphology. Additionally, it was shown that concentrations of ammonium and total nitrogen correlated strongly to the presence of foam-formers. This study provided new insight into the identity of putative foam-forming microorganisms in mesophilic AD systems, allowing for the subsequent surveillance of their abundances and studies of their ecology. Such information will importantly inform the development of control measures for these problematic microorganisms

A comprehensive overview of the Chloroflexota community in wastewater treatment plants worldwide

Filamentous Chloroflexota are abundant in activated sludge wastewater treatment plants (WWTPs) worldwide and are occasionally associated with poor solid-liquid separation or foaming, but most of the abundant lineages remain undescribed. Here, we present a comprehensive overview of Chloroflexota abundant in WWTPs worldwide, using high-quality metagenome-assembled genomes (MAGs) and 16S rRNA amplicon data from 740 Danish and global WWTPs. Many novel taxa were described, encompassing 4 families, 13 genera, and 29 novel species. These were widely distributed across most continents, influenced by factors such as climate zone and WWTP process design. Visualization by fluorescence in situ hybridization (FISH) confirmed their high abundances in many WWTPs based on the amplicon data and showed a filamentous morphology for nearly all species. Most formed thin and short trichomes integrated into the floc structure, unlikely to form the typical inter-floc bridging that hinders activated sludge floc settling. Metabolic reconstruction of 53 high-quality MAGs, representing most of the novel genera, offered further insights into their versatile metabolisms and suggested a primary role in carbon removal and involvement in nitrogen cycling. The presence of glycogen reserves, detected by FISH-Raman microspectroscopy, seemed widespread across the phylum, demonstrating that these bacteria likely utilize glycogen as energy storage to survive periods with limited resources. This study gives a broad overview of the Chloroflexota community in global activated sludge WWTPs and improves our understanding of their roles in these engineered ecosystems.</p