The novel genus, ‘Candidatus Phosphoribacter’, previously identified as Tetrasphaera, is the dominant polyphosphate accumulating lineage in EBPR wastewater treatment plants worldwide

The bacterial genus Tetrasphaera encompasses abundant polyphosphate accumulating organisms (PAOs) that are responsible for enhanced biological phosphorus removal (EBPR) in wastewater treatment plants. Recent analyses of genomes from pure cultures revealed that 16S rRNA genes cannot resolve the lineage, and that Tetrasphaera spp. are from several different genera within the Dermatophilaceae. Here, we examine 14 recently recovered high-quality metagenome-assembled genomes from wastewater treatment plants containing full-length 16S rRNA genes identified as Tetrasphaera, 11 of which belong to the uncultured Tetrasphaera clade 3. We find that this clade represents two distinct genera, named here Ca. Phosphoribacter and Ca. Lutibacillus, and reveal that the widely used model organism Tetrasphaera elongata is less relevant for physiological predictions of this uncultured group. Ca. Phosphoribacter incorporates species diversity unresolved at the 16S rRNA gene level, with the two most abundant and often co-occurring species encoding identical V1-V3 16S rRNA gene amplicon sequence variants but different metabolic capabilities, and possibly, niches. Both Ca. P. hodrii and Ca. P. baldrii were visualised using fluorescence in situ hybridisation (FISH), and PAO capabilities were confirmed with FISH-Raman microspectroscopy and phosphate cycling experiments. Ca. Phosphoribacter represents the most abundant former Tetrasphaera lineage and PAO in EPBR systems in Denmark and globally

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

MiDAS 4: A global catalogue of full-length 16S rRNA gene sequences and taxonomy for studies of bacterial communities in wastewater treatment plants

Microbial communities are responsible for biological wastewater treatment, but our knowledge of their diversity and function is still poor. Here, we sequence more than 5 million high-quality, full-length 16S rRNA gene sequences from 740 wastewater treatment plants (WWTPs) across the world and use the sequences to construct the ‘MiDAS 4’ database. MiDAS 4 is an amplicon sequence variant resolved, full-length 16S rRNA gene reference database with a comprehensive taxonomy from domain to species level for all sequences. We use an independent dataset (269 WWTPs) to show that MiDAS 4, compared to commonly used universal reference databases, provides a better coverage for WWTP bacteria and an improved rate of genus and species level classification. Taking advantage of MiDAS 4, we carry out an amplicon-based, global-scale microbial community profiling of activated sludge plants using two common sets of primers targeting regions of the 16S rRNA gene, revealing how environmental conditions and biogeography shape the activated sludge microbiota. We also identify core and conditionally rare or abundant taxa, encompassing 966 genera and 1530 species that represent approximately 80% and 50% of the accumulated read abundance, respectively. Finally, we show that for well-studied functional guilds, such as nitrifiers or polyphosphate-accumulating organisms, the same genera are prevalent worldwide, with only a few abundant species in each genus

MiDAS 4: A global catalogue of full-length 16S rRNA gene sequences and taxonomy for studies of bacterial communities in wastewater treatment plants

Microbial communities are responsible for biological wastewater treatment, but our knowledge of their diversity and function is still poor. Here, we sequence more than 5 million high-quality, full-length 16S rRNA gene sequences from 740 wastewater treatment plants (WWTPs) across the world and use the sequences to construct the ‘MiDAS 4’ database. MiDAS 4 is an amplicon sequence variant resolved, full-length 16S rRNA gene reference database with a comprehensive taxonomy from domain to species level for all sequences. We use an independent dataset (269 WWTPs) to show that MiDAS 4, compared to commonly used universal reference databases, provides a better coverage for WWTP bacteria and an improved rate of genus and species level classification. Taking advantage of MiDAS 4, we carry out an amplicon-based, global-scale microbial community profiling of activated sludge plants using two common sets of primers targeting regions of the 16S rRNA gene, revealing how environmental conditions and biogeography shape the activated sludge microbiota. We also identify core and conditionally rare or abundant taxa, encompassing 966 genera and 1530 species that represent approximately 80% and 50% of the accumulated read abundance, respectively. Finally, we show that for well-studied functional guilds, such as nitrifiers or polyphosphate-accumulating organisms, the same genera are prevalent worldwide, with only a few abundant species in each genus.Fil: Dueholm, Morten Kam Dahl. Aalborg University; DinamarcaFil: Nierychlo, Marta. Aalborg University; DinamarcaFil: Andersen, Kasper Skytte. Aalborg University; DinamarcaFil: Rudkjøbing, Vibeke. Aalborg University; DinamarcaFil: Knutsson, Simon. Aalborg University; DinamarcaFil: Arriaga, Sonia. Instituto Potosino de Investigación Científica y Tecnológica; MéxicoFil: Bakke, Rune. University College of Southeast Norway; NoruegaFil: Boon, Nico. University of Ghent; BélgicaFil: Bux, Faizal. Durban University of Technology; SudáfricaFil: Christensson, Magnus. Veolia Water Technologies Ab; SueciaFil: Chua, Adeline Seak May. University Malaya; MalasiaFil: Curtis, Thomas P.. University of Newcastle; Reino UnidoFil: Cytryn, Eddie. Agricultural Research Organization Of Israel; IsraelFil: Erijman, Leonardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires; ArgentinaFil: Etchebehere, Claudia. Instituto de Investigaciones Biológicas "Clemente Estable"; UruguayFil: Fatta Kassinos, Despo. University of Cyprus; ChipreFil: Frigon, Dominic. McGill University; CanadáFil: Garcia Chaves, Maria Carolina. Universidad de Antioquia; ColombiaFil: Gu, April Z.. Cornell University; Estados UnidosFil: Horn, Harald. Karlsruher Institut Für Technologie; AlemaniaFil: Jenkins, David. David Jenkins & Associates Inc; Estados UnidosFil: Kreuzinger, Norbert. Tu Wien; AustriaFil: Kumari, Sheena. Durban University of Technology; SudáfricaFil: Lanham, Ana. University of Bath; Reino UnidoFil: Law, Yingyu. Singapore Centre For Environmental Life Sciences Engineering; SingapurFil: Leiknes, TorOve. King Abdullah University of Science and Technology; Arabia SauditaFil: Morgenroth, Eberhard. Eth Zürich; SuizaFil: Muszyński, Adam. Politechnika Warszawska; PoloniaFil: Petrovski, Steve. La Trobe University; AustraliaFil: Pijuan, Maite. Catalan Institute For Water Research; EspañaFil: Pillai, Suraj Babu. Va Tech Wabag Ltd; IndiaFil: Reis, Maria A. M.. Universidade Nova de Lisboa; PortugalFil: Rong, Qi. Chinese Academy of Sciences; ChinaFil: Rossetti, Simona. Istituto Di Ricerca Sulle Acque (irsa) ; Consiglio Nazionale Delle Ricerche;Fil: Seviour, Robert. La Trobe University; AustraliaFil: Tooker, Nick. University of Massachussets; Estados UnidosFil: Vainio, Pirjo. Espoo R&D Center; FinlandiaFil: van Loosdrecht, Mark. Delft University of Technology; Países BajosFil: Vikraman, R.. VA Tech Wabag, Philippines Inc; FilipinasFil: Wanner, Jiří. University of Chemistry And Technology; República ChecaFil: Weissbrodt, David. Delft University of Technology; Países BajosFil: Wen, Xianghua. Tsinghua University; ChinaFil: Zhang, Tong. The University of Hong Kong; Hong KongFil: Nielsen, Per H.. Aalborg University; DinamarcaFil: Albertsen, Mads. Aalborg University; DinamarcaFil: Nielsen, Per Halkjær. Aalborg University; Dinamarc

Genomic insights into members of the candidate phylum Hyd24-12 common in mesophilic anaerobic digesters

Members of the candidate phylum Hyd24-12 are globally distributed, but no genomic information or knowledge about their morphology, physiology or ecology is available. In this study, members of the Hyd24-12 lineage were shown to be present and abundant in full-scale mesophilic anaerobic digesters at Danish wastewater treatment facilities. In some samples, a member of the Hyd24-12 lineage was one of the most abundant genus-level bacterial taxa, accounting for up to 8% of the bacterial biomass. Three closely related and near-complete genomes were retrieved using metagenome sequencing of full-scale anaerobic digesters. Genome annotation and metabolic reconstruction showed that they are Gram-negative bacteria likely involved in acidogenesis, producing acetate and hydrogen from fermentation of sugars, and may play a role in the cycling of sulphur in the digesters. Fluorescence in situ hybridization revealed single rod-shaped cells dispersed within the flocs. The genomic information forms a foundation for a more detailed understanding of their role in anaerobic digestion and provides the first insight into a hitherto undescribed branch in the tree of life

Nonlocality of a QCD vacuum and the ALEPH data on tau lepton decay

Settleability of particles in activated sludge systems can be impaired by an overgrowth of filamentous bacteria, a problem known as bulking. These filaments are often members of the phylum Chloroflexi, sometimes reaching abundances in excess of 30% of the biovolume. The uncultured Chloroflexi phylotype, Candidatus Amarolinea, has been observed in high abundances in Danish full-scale activated sludge systems by 16S rRNA gene amplicon surveys, where it has been associated with bulking. In this study, fluorescence in situ hybridization was applied to confirm their high abundance, filamentous morphology, and contribution to the interfloc bridging that characterizes filamentous bulking. Furthermore, genome-centric metagenomics using both Illumina and Oxford Nanopore sequencing was used to obtain a near complete population genome (5.7 Mbp) of the Ca. Amarolinea phylotype, which belongs to the proposed novel family Amarolineaceae within the order Caldilineales of Chloroflexi. Annotation of the genome indicated that the phylotype is capable of aerobic respiration, fermentation, and dissimilatory nitrate reduction to ammonia. The genome sequence also gives a better insight into the phylogenetic and evolutionary relationships of the organism. The name Candidatus Amarolinea aalborgensis is proposed for the species

Identification of active denitrifiers in full-scale nutrient removal wastewater treatment systems

Denitrification is essential to the removal of nitrogen from wastewater during treatment, yet an understanding of the diversity of the active denitrifying bacteria responsible in full‐scale wastewater treatment plants (WWTPs) is lacking. In this study, stable‐isotope probing (SIP) was applied in combination with microautoradiography (MAR)‐fluorescence in situ hybridization (FISH) to identify previously unrecognized active denitrifying phylotypes in a full‐scale WWTP with biological N and P removal. Acknowledging that different denitrifiers will have specific carbon source preferences, a fully 13C‐labelled complex substrate was used for SIP incubations, under nitrite‐reducing conditions, in order to maximize the capture of the potentially metabolically diverse denitrifiers likely present. Members of the Rhodoferax, Dechloromonas, Sulfuritalea, Haliangium and Thermomonas were represented in the 16S rRNA gene clone libraries from DNA enriched in 13C, with FISH probes optimized here for their in situ characterization. FISH and MAR confirmed that they were all active denitrifiers in the community. The combined approach of SIP and MAR‐FISH represents an excellent approach for identifying and characterizing an un‐described diversity of active denitrifiers in full‐scale systems

The impact of immigration on microbial community composition in full-scale anaerobic digesters

Anaerobic digestion is widely applied to treat organic waste at wastewater treatment plants. Characterisation of the underlying microbiology represents a source of information to develop strategies for improved operation. Hence, we investigated microbial communities of thirty-two full-scale anaerobic digesters over a six-year period using 16S rRNA gene amplicon sequencing. Sampling of the sludge fed into these systems revealed that several of the most abundant populations were likely inactive and immigrating with the influent. This observation indicates that a failure to consider immigration will interfere with correlation analysis and give an inaccurate picture of the growing microbial community. Furthermore, several abundant OTUs could not be classified to genus level with commonly applied taxonomies, making inference of their function unreliable and comparison to other studies problematic. As such, the existing MiDAS taxonomy was updated to include these abundant phylotypes. The communities of individual digesters surveyed were remarkably similar - with only 300 OTUs representing 80% of the total reads across all plants, and 15% of these identified as non-growing and possibly inactive immigrating microbes. By identifying abundant and growing taxa in anaerobic digestion, this study paves the way for targeted characterisation of the process-important organisms towards an in-depth understanding of the microbiology

Long-term effects of sulphide on the enhanced biological removal of phosphorus: The symbiotic role of Thiothrix caldifontis

Thiothrix caldifontis was the dominant microorganism (with an estimated bio-volume of 65 ± 3%) in a lab-scale enhanced biological phosphorus removal (EBPR) system containing 100 mg of sulphide per litre in the influent. After a gradual exposure to the presence of sulphide, the EBPR system initially dominated by Candidatus Accumulibacter phosphatis Clade I (98 ± 3% bio-volume) (a known polyphosphate accumulating organism, PAO) became enriched with T. caldifontis. Throughout the different operating conditions studied, practically 100% phosphate removal was always achieved. The gradual increase of the sulphide content in the medium (added to the anaerobic stage of the alternating anaerobic-aerobic sequencing batch reactor) and the adjustment of the aerobic hydraulic retention time played a major role in the enrichment of T. caldifontis. T. caldifontis exhibited a mixotrophic metabolism by storing carbon anaerobically as poly-β-hydroxy-alkanoates (PHA) and generating the required energy through the hydrolysis of polyphosphate. PHA was used in the aerobic period as carbon and energy source for growth, polyphosphate, and glycogen formation. Apparently, extra energy was obtained by the initial accumulation of sulphide as an intracellular sulphur, followed by its gradual oxidation to sulphate. The culture enriched with T. caldifontis was able to store approximately 100 mg P/g VSS. This research suggests that T. caldifontis could behave like PAO with a mixotrophic metabolism for phosphorus removal using an intracellular sulphur pool as energy source. These findings can be of major interest for the biological removal of phosphorus from wastewaters with low organic carbon concentrations containing reduced S-compounds like those (pre-)treated in anaerobic systems or from anaerobic sewers