mSystems

ABSTRACT Polyphosphate-accumulating organisms (PAOs) are the main bacteria responsible for phosphorus removal and recovery in full-scale wastewater treatment plants (WWTPs). They encompass members of the genera Candidatus Accumulibacter, Azonexus (formerly Dechloromonas ), and Candidatus Phosphoribacter (formerly Tetrasphaera ), with most studies focusing on Ca . Accumulibacter, primarily using lab-scale enrichment cultures. Although members from the three genera often co-exist in full-scale WWTPs, the metabolic capabilities and traits that determine the niche differentiation of the specific species are still unknown. We retrieved 214 high-quality metagenome-assembled genomes from a full-scale plant with phosphorus removal and examined the polyphosphate-related metabolic pathways using genome-resolved metatranscriptomics in the different process tanks in situ and by using short-term incubations ex situ . We observed the co-existence of nine uncultured PAO species from the three genera with clear niche differentiation in the utilization of different carbon sources and involvement in the denitrification process. Additionally, we observed several physiological differences among species of the same genus, indicating variations in niche specialization. This suggests that biological P removal and other processes in full-scale WWTPs are carried out by a complex and diverse PAO community that together ensures stable plant performance. IMPORTANCE The current understanding of the ecology and physiology of polyphosphate-accumulating organisms (PAOs) is mostly based on Candidatus Accumulibacter, primarily studied in enriched lab-scale studies. Recent taxonomic reclassification revealed that the most studied Ca . Accumulibacter species are either not present or present in low abundance in full-scale wastewater treatment plants (WWTPs). This raises concerns that knowledge from lab-scale studies may not apply to species in full-scale plants. Additionally, the indication of a distinct PAO physiology in Candidatus Phosphoribacter compared to Ca . Accumulibacter and the other abundant PAO Ca . Azonexus poses further questions about the accuracy of the current PAO model. Here, we show that in full-scale plant species from Ca . Accumulibacter, Ca . Azonexus, and Ca . Phosphoribacter always co-exist, and they have distinct niche separations in terms of carbon source utilization and the use of electron acceptors. This co-existence and metabolic diversity indicate that a complex microbial community is crucial for efficient phosphorus removal in full-scale WWTPs.

ABSTRACT Ca . Babelota is a phylum of strictly intracellular bacteria whose representatives are commonly detected in various environments through metagenomics, though their presence, ecology, and biology have never been addressed so far. As a group of strict intracellular, we hypothesize that their presence, occurrence, and abundance heavily depend on their hosts, which are known as heterotrophic protists, based on few described isolates. Here, we conducted a sampling campaign allowing to characterize protists and associated bacterial communities, using high-throughput sequencing. In parallel, a systematic enrichment of protists from samples was performed to attempt characterization and isolation of new Ca . Babelota within native hosts. We found that Ca . Babelota are among the most widespread phylum among the rare ones. Protist enrichments are allowed in certain cases to enrich as well for Ca . Babelota, which could be visualized in vivo infecting protist cells. Though cosmopolitan, Ca . Babelota diversity was highly site-specific. Cooccurrence analyses allowed to retrieve well-known as well as new putative associations involving numerous protists of various trophic regimes. The combination of approaches developed in this study enhances our understanding of Ca . Babelota ecology and biology, while paving the way for future isolation of new members of this elusive phylum, which could have huge impact on protists—and ecosystems—functioning. IMPORTANCE Our understanding of microbial diversity surrounding us and colonizing the environment has been dramatically impacted by the advent of DNA-based analyses. Such progress helped shine a new light on numerous lineages of yet-to-be-characterized microbes, whose ecology and biology are basically unknown. Among those uncharacterized clades is the Candidatus Babelota, a bacterial phylum for which parasitism seems to be an ancestral trait. All known Ca. Babelota thrive by infecting phagotrophic protist hosts, thereby impacting this basal link of the trophic chain. The Ca. Babelota constitutes a model that stands out, as phylum-wide conserved parasitism has only been described in one previous occurrence for Bacteria, with the Chlamydiota. Thus, exploring the intricate interplay between Ca. Babelota and their protist hosts will advance our knowledge of bacterial diversity, their ecology, and global impact on ecosystem functioning.

ABSTRACT The phylum Gemmatimonadota is widespread but rarely cultured and, in fact, there are only six described species isolated from soil, freshwater, and wastewater treatment. However, no isolates of Gemmatimonadota from marine environment have been described; thus, little is known about the physiology and metabolism of members of the marine lineages. In this study, four novel facultatively anaerobic bacterial strains belonging to Gemmatimonadota were isolated from marine sediments collected from Xiaoshi Island in Weihai, China, using an aerobic enrichment method. The integrated results of phylogenetic and phenotypic characteristics supported that these four strains represent one novel species in a novel genus, for which the name Gaopeijia maritima gen. nov., sp. nov. is proposed, as the first representative of novel taxa, Gaopeijiales ord. nov., Gaopeijiaceae fam. nov. in the class Longimicrobiia. Gaopeijiales was detected in 22,884 out of 95,549 amplicon data sets, mainly from soil. However, the highest mean relative abundances were in sponge (0.7%) and marine sediment (0.35%), showing salt-related character. Most of the Gaopeijiales subgroups potentially belong to the rare bacterial biosphere. The aerobic enrichment in this study could significantly increase the relative abundance of Gaopeijiales (from 0.37% to 2.6%). Furthermore, the metabolic capabilities inferred from high-quality representative Gaopeijiales genomes/MAGs suggest that this group primarily performs chemoorganoheterotrophic metabolism with facultatively anaerobic characteristics and possesses various secondary metabolite biosynthesis gene clusters (BGCs), mirroring those observed in the four novel strains. IMPORTANCE Despite rapid advances in molecular and sequencing technologies, obtaining pure cultures remains a crucial research goal in microbiology, as it is essential for a deeper understanding of microbial metabolism. Gemmatimonadota is a widespread but rarely cultured bacterial phylum. Currently, there are only six cultured strains of this interesting group, all isolated from non-marine environments. Little is known about the physiology and metabolism of members of the marine lineages. Here we isolated and characterized four novel marine strains, and proposed a new order Gaopeijiales within Gemmatimonadota . Furthermore, the global distribution, environmental preference, and metabolic potential of Gaopeijiales are analyzed using public data. Our work enriches the resources available for the under-represented phylum Gemmatimonadota and provides insights into the physiological and metabolic characteristics of the marine lineage ( Gaopeijiales ) through culturology and omics.

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.

The discovery of the new halophilic archaeon, Halorutilus salinus , representing a novel order, family, genus, and species within the class Halobacteria and phylum Euryarchaeota clearly enables insights into the microbial dark matter, expanding the current taxonomical knowledge of this group of archaea. The in-depth comparative genomic analysis performed on this new taxon revealed one of the first known examples of an Halobacteria representative coding the archaeal RuBisCO gene and with a streamlined genome, being ecologically successful in nature and explaining its previous non-isolation.