Nielsen, Per H.

ABSTRACT 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 filamentous bulking 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 fluorescence in 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. IMPORTANCE The genus Leptothrix has been extensively studied and described since the 1880s, with six species currently described but with the majority uncultured and undescribed. Some species are assumed to have a filamentous morphology and can cause settling problems in wastewater treatment plants (WWTPs). Here, we revised the classification of the most abundant Leptothrix spp. present in WWTPs across the world, showing that most belong to other genera, such as Rubrivivax and Ideonella . Furthermore, most do not have a filamentous morphology and are not problematic in WWTPs as previously believed. Metabolic reconstruction, including some traits validated in situ by the application of new fluorescence in situ hybridization probes and Raman microspectroscopy, provided additional insights into their metabolism. The study has contributed to a better understanding of the diversity, morphology, and function of “ Leptothrix ,” which belong to the abundant core community across global activated sludge WWTPs.

ABSTRACT 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. IMPORTANCE Chloroflexota are often abundant members of the biomass in wastewater treatment plants (WWTPs) worldwide, typically with a filamentous morphology, forming the backbones of the activated sludge floc. However, their overgrowth can often cause operational issues connected to poor settling or foaming, impairing effluent quality and increasing operational costs. Despite their importance, few Chloroflexota genera have been characterized so far. Here, we present a comprehensive overview of Chloroflexota abundant in WWTPs worldwide and an in-depth characterization of their morphology, phylogeny, and ecophysiology, obtaining a broad understanding of their ecological role in activated sludge.

AbstractCandidatusAccumulibacter is a key genus of polyphosphate-accumulating organisms (PAOs) found in laboratory- and full-scale wastewater treatment systems, mediating enhanced biological phosphorus removal (EBPR). However, little is known about their ability to resist phage infection. We conducted a systematic analysis of the occurrence and characteristics of clustered regularly interspaced short palindromic repeats and associated proteins (CRISPR-Cas) systems and prophages in diverseCa.Accumulibacter taxa (43 in total, including 10 newly recovered genomes). Fourty complete CRISPR loci were identified in 28 genomes, falling into 6 subtypes. The occurrence and distribution of CRISPR-Cas systems did not follow a vertical evolutionary relationship. Phylogenetic analyses of thecasgenes and direct repeats (DRs) suggested that the CRISPR-Cas systems were likely acquired via horizontal gene transfer, with acquisition rates higher than those of speciation, rendering differentCa.Accumulibacter distinct adaptivity to phage predations. 2448 spacers were identified, 67 of them matched to known phages. Major differences were observed among the numbers of spacers for differentCa.Accumulibacter, showing unique phages that could be resisted by different members. A comparison of the spacers in genomes having the samecasgene but from distinct geographical locations indicated that habitat isolation may have resulted in the acquisition of different spacers by differentCa. Accumulibacter. Metagenomic analysis allowed the identification of 26 viral contigs (18 are Caudovirales members) in 6 metagenomic datasets from three lab-scale enrichment reactors, matching to 73 spacers in 10Ca.Accumulibacter genomes in these reactors, showing the specific immunity of theseCa.Accumulibacter. Metatranscriptomic analyses showed the activity of the CRISPR-Cas system under both anaerobic and aerobic conditions. Extra efforts were made to identify prophages inCa.Accumulibacter genomes. In total, 133 prophage regions were identified. Twenty-seven of them were classified as potentially active prophages. Three prophages (all are Caudovirales members, in DS2011, SCELSE-7IIH and SCELSE-5IIH, respectively) are readily activable. Differences in the occurrence of CRISPR-Cas systems and prophages inCa.Accumulibacter will lead to their distinct responses under phage predation. This study represents the first systematic analysis of CRISPR-Cas systems and prophages with combined experimental and bioinformatic methods in theCa.Accumulibacter lineage, providing new perspectives on the potential impacts of phages onCa.Accumulibacter and EBPR systems.

AbstractCandidatusAccumulibacter was the first microorganism identified as a polyphosphate-accumulating organism (PAO), important for phosphorus removal from wastewater. This genus is diverse, and the current phylogeny and taxonomic framework appears complicated, with the majority of publicly available genomes classified as “CandidatusAccumulibacter phosphatis”, despite notable phylogenetic divergence. Theppk1marker gene allows for a finer scale differentiation into different “types” and “clades”, nevertheless taxonomic assignments remain confusing and inconsistent across studies. Therefore, a comprehensive re-evaluation is needed to establish a common understanding of this genus, both in terms of naming and basic conserved physiological traits. Here, we provide this re-assessment using a comparison of genome,ppk1, and 16S rRNA gene-based approaches from comprehensive datasets. We identified 15 novel species, along with the well-knownCa. A. phosphatis,Ca. A. deltensis andCa. A. aalborgensis. To compare the speciesin situ, we designed new species-specific FISH probes and revealed their morphology and arrangement in activated sludge. Based on the MiDAS global survey,Ca. Accumulibacter species were widespread in WWTPs with phosphorus removal, indicating the process design as a major driver for their abundance. Genome mining for PAO related pathways and FISH-Raman microspectroscopy confirmed the potential for the PAO metabolism in allCa. Accumulibacter species, with detectionin situof the typical PAO storage polymers. Genome annotation further revealed fine-scale 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.ImportanceCandidatusAccumulibacter is the most studied PAO organism, 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. 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 16S rRNA gene- and other phylogenetic markers phylogeny, we obtained a higher resolution taxonomy and established a common understanding of this genus. Furthermore, genome mining of gene and pathways of interest, validatedin situby application of a new set of FISH probes and Raman micromicrospectroscopy, provided additional high-resolution metabolic insights into these organisms.

CandidatusMicrothrix 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 investigateCa.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 dominantCa.M. parvicella and an unknown species typically present along withCa.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 “CandidatusMicrothrix subdominans.” Ten high-quality metagenome-assembled genomes recovered from Danish WWTPs, including 6 representing the novelCa.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 fluorescencein situhybridization probes revealed similar filamentous morphology for both species. Raman microspectroscopy was used to quantify thein situlevels of intracellular poly-P. Despite the observed similarities in their physiology (both by genomes andin 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 intoin situgene expression and regulation, while the new FISH probes provide a useful tool for further characterizationin situ. This study is an important step toward understanding the ecology ofCa.Microthrix in WWTPs, which may eventually lead to optimization of control strategies for its growth in this ecosystem.

AbstractMembers of the genusDechloromonasare often abundant in enhanced biological phosphorus removal (EBPR) systems and are recognized putative polyphosphate accumulating organisms (PAOs), but their role in phosphate (P) removal is still unclear. Here, we used 16S rRNA gene sequencing and fluorescencein situhybridization (FISH) to investigate the abundance and distribution ofDechloromonasspp. in Danish wastewater treatment plants. Two species were abundant, novel, and uncultured, and could be targeted by existing FISH probes. Raman microspectroscopy of probe-defined organisms (FISH-Raman) revealed the levels and dynamics of important intracellular storage polymers in abundantDechloromonasspp. in the activated sludge from four full-scale EBPR plants and from a lab-scale sequencing batch reactor fed with different carbon sources (acetate, glucose, glycine, and glutamate). Moreover, 7 distinctDechloromonasspecies were determined from a set of 10 high-quality metagenome-assembled genomes (MAGs) from Danish EBPR plants, each encoding the potential for poly-P, glycogen, and polyhydroxyalkanoates (PHA) accumulation. The two most abundant species exhibited anin situphenotype in complete accordance with the metabolic information retrieved by the MAGs, with dynamic levels of poly-P, glycogen, and PHA during feast-famine anaerobic-aerobic cycling, legitimately placing these microorganisms among the important PAOs. As no isolates are available for the two species, we propose the namesCandidatusDechloromonas phosphatis andCandidatusDechloromonas phosphovora.