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ABSTRACT Members of the “ Candidatus Accumulibacter” genus are widely studied as key polyphosphate-accumulating organisms (PAOs) in biological nutrient removal (BNR) facilities performing enhanced biological phosphorus removal (EBPR). This diverse lineage includes 18 “ Ca . Accumulibacter” species, which have been proposed based on the phylogenetic divergence of the polyphosphate kinase 1 ( ppk1 ) gene and genome-scale comparisons of metagenome-assembled genomes (MAGs). Phylogenetic classification based on the 16S rRNA genetic marker has been difficult to attain because most “ Ca . Accumulibacter” MAGs are incomplete and often do not include the rRNA operon. Here, we investigate the “ Ca . Accumulibacter” diversity in pilot-scale treatment trains performing BNR under low dissolved oxygen (DO) conditions using genome-resolved metagenomics. Using long-read sequencing, we recovered medium- and high-quality MAGs for 5 of the 18 “ Ca . Accumulibacter” species, all with rRNA operons assembled, which allowed a reassessment of the 16S rRNA-based phylogeny of this genus and an analysis of phylogeny based on the 23S rRNA gene. In addition, we recovered a cluster of MAGs that based on 16S rRNA, 23S rRNA, ppk1 , and genome-scale phylogenetic analyses do not belong to any of the currently recognized “ Ca . Accumulibacter” species for which we propose the new species designation “ Ca . Accumulibacter jenkinsii” sp. nov. Relative abundance evaluations of the genus across all pilot plant operations revealed that regardless of the operational mode, “ Ca . A. necessarius” and “ Ca . A. propinquus” accounted for more than 40% of the “ Ca . Accumulibacter” community, whereas the newly proposed “ Ca . A. jenkinsii” accounted for about 5% of the “ Ca . Accumulibacter” community. IMPORTANCE One of the main drivers of energy use and operational costs in activated sludge processes is the amount of oxygen provided to enable biological phosphorus and nitrogen removal. Wastewater treatment facilities are increasingly considering reduced aeration to decrease energy consumption, and whereas successful BNR has been demonstrated in systems with minimal aeration, an adequate understanding of the microbial communities that facilitate nutrient removal under these conditions is still lacking. In this study, we used genome-resolved metagenomics to evaluate the diversity of the “ Candidatus Accumulibacter” genus in pilot-scale plants operating with minimal aeration. We identified the “ Ca . Accumulibacter” species enriched under these conditions, including one novel species for which we propose “ Ca . Accumulibacter jenkinsii” sp. nov. as its designation. Furthermore, the MAGs obtained for five additional “ Ca . Accumulibacter” species further refine the phylogeny of the “ Ca . Accumulibacter” genus and provide new insight into its diversity within unconventional biological nutrient removal systems.

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.

ABSTRACT The phylum Gemmatimonadota comprises mainly uncultured microorganisms that inhabit different environments such as soils, freshwater lakes, marine sediments, sponges, or corals. Based on 16S rRNA gene studies, the group PAUC43f is one of the most frequently retrieved Gemmatimonadota in marine samples. However, its physiology and ecological roles are completely unknown since, to date, not a single PAUC43f isolate or metagenome-assembled genome (MAG) has been characterized. Here, we carried out a broad study of the distribution, abundance, ecotaxonomy, and metabolism of PAUC43f, for which we propose the name of Palauibacterales . This group was detected in 4,965 16S rRNA gene amplicon datasets, mainly from marine sediments, sponges, corals, soils, and lakes, reaching up to 34.3% relative abundance, which highlights its cosmopolitan character, mainly salt-related. The potential metabolic capabilities inferred from 52 Palauibacterales MAGs recovered from marine sediments, sponges, and saline soils suggested a facultative aerobic and chemoorganotrophic metabolism, although some members may also oxidize hydrogen. Some Palauibacterales species might also play an environmental role as N 2 O consumers as well as suppliers of serine and thiamine. When compared to the rest of the Gemmatimonadota phylum, the biosynthesis of thiamine was one of the key features of the Palauibacterales . Finally, we show that polysaccharide utilization loci (PUL) are widely distributed within the Gemmatimonadota so that they are not restricted to Bacteroidetes , as previously thought. Our results expand the knowledge about this cryptic phylum and provide new insights into the ecological roles of the Gemmatimonadota in the environment. IMPORTANCE Despite advances in molecular and sequencing techniques, there is still a plethora of unknown microorganisms with a relevant ecological role. In the last years, the mostly uncultured Gemmatimonadota phylum is attracting scientific interest because of its widespread distribution and abundance, but very little is known about its ecological role in the marine ecosystem. Here we analyze the global distribution and potential metabolism of the marine Gemmatimonadota group PAUC43f, for which we propose the name of Palauibacterales order. This group presents a saline-related character and a chemoorganoheterotrophic and facultatively aerobic metabolism, although some species might oxidize H 2 . Given that Palauibacterales is potentially able to synthesize thiamine, whose auxotrophy is the second most common in the marine environment, we propose Palauibacterales as a key thiamine supplier to the marine communities. This finding suggests that Gemmatimonadota could have a more relevant role in the marine environment than previously thought.

Recent advances in sequencing technology promoted the blowout discovery of super tiny microbes in the Diapherotrites , Parvarchaeota , Aenigmarchaeota , Nanoarchaeota , and Nanohaloarchaeota (DPANN) superphylum. However, the unculturable properties of the majority of microbes impeded our investigation of their behavior and symbiotic lifestyle in the corresponding community.