Oxford Nanopore R10.4 long-read sequencing enables the generation of near-finished bacterial genomes from pure cultures and metagenomes without short-read or reference polishing.

Long-read Oxford Nanopore sequencing has democratized microbial genome sequencing and enables the recovery of highly contiguous microbial genomes from isolates or metagenomes. However, to obtain near-finished genomes it has been necessary to include short-read polishing to correct insertions and deletions derived from homopolymer regions. Here, we show that Oxford Nanopore R10.4 can be used to generate near-finished microbial genomes from isolates or metagenomes without short-read or reference polishing.

High-throughput DNA extraction and cost-effective miniaturized metagenome and amplicon library preparation of soil samples for DNA sequencing.

Reductions in sequencing costs have enabled widespread use of shotgun metagenomics and amplicon sequencing, which have drastically improved our understanding of the microbial world. However, large sequencing projects are now hampered by the cost of library preparation and low sample throughput, comparatively to the actual sequencing costs. Here, we benchmarked three high-throughput DNA extraction methods: ZymoBIOMICS™ 96 MagBead DNA Kit, MP BiomedicalsTM FastDNATM-96 Soil Microbe DNA Kit, and DNeasy® 96 PowerSoil® Pro QIAcube® HT Kit. The DNA extractions were evaluated based on length, quality, quantity, and the observed microbial community across five diverse soil types. DNA extraction of all soil types was successful for all kits, however DNeasy® 96 PowerSoil® Pro QIAcube® HT Kit excelled across all performance parameters. We further used the nanoliter dispensing system I.DOT One to miniaturize Illumina amplicon and metagenomic library preparation volumes by a factor of 5 and 10, respectively, with no significant impact on the observed microbial communities. With these protocols, DNA extraction, metagenomic, or amplicon library preparation for one 96-well plate are approx. 3, 5, and 6 hours, respectively. Furthermore, the miniaturization of amplicon and metagenome library preparation reduces the chemical and plastic costs from 5.0 to 3.6 and 59 to 7.3 USD pr. sample. This enhanced efficiency and cost-effectiveness will enable researchers to undertake studies with greater sample sizes and diversity, thereby providing a richer, more detailed view of microbial communities and their dynamics.

Generation of Comprehensive Ecosystem-Specific Reference Databases with Species-Level Resolution by High-Throughput Full-Length 16S rRNA Gene Sequencing and Automated Taxonomy Assignment (AutoTax).

High-throughput 16S rRNA gene amplicon sequencing is an essential method for studying the diversity and dynamics of microbial communities. However, this method is presently hampered by the lack of high-identity reference sequences for many environmental microbes in the public 16S rRNA gene reference databases and by the absence of a systematic and comprehensive taxonomy for the uncultured majority. Here, we demonstrate how high-throughput synthetic long-read sequencing can be applied to create ecosystem-specific full-length 16S rRNA gene amplicon sequence variant (FL-ASV) resolved reference databases that include high-identity references (>98.7% identity) for nearly all abundant bacteria (>0.01% relative abundance) using Danish wastewater treatment systems and anaerobic digesters as an example. In addition, we introduce a novel sequence identity-based approach for automated taxonomy assignment (AutoTax) that provides a complete seven-rank taxonomy for all reference sequences, using the SILVA taxonomy as a backbone, with stable placeholder names for unclassified taxa. The FL-ASVs are perfectly suited for the evaluation of taxonomic resolution and bias associated with primers commonly used for amplicon sequencing, allowing researchers to choose those that are ideal for their ecosystem. Reference databases processed with AutoTax greatly improves the classification of short-read 16S rRNA ASVs at the genus- and species-level, compared with the commonly used universal reference databases. Importantly, the placeholder names provide a way to explore the unclassified environmental taxa at different taxonomic ranks, which in combination with in situ analyses can be used to uncover their ecological roles.

Novel prosthecate bacteria from the candidate phylum Acetothermia.

Members of the candidate phylum Acetothermia are globally distributed and detected in various habitats. However, little is known about their physiology and ecological importance. In this study, an operational taxonomic unit belonging to Acetothermia was detected at high abundance in four full-scale anaerobic digesters by 16S rRNA gene amplicon sequencing. The first closed genome from this phylum was obtained by differential coverage binning of metagenomes and scaffolding with long nanopore reads. Genome annotation and metabolic reconstruction suggested an anaerobic chemoheterotrophic lifestyle in which the bacterium obtains energy and carbon via fermentation of peptides, amino acids, and simple sugars to acetate, formate, and hydrogen. The morphology was unusual and composed of a central rod-shaped cell with bipolar prosthecae as revealed by fluorescence in situ hybridization combined with confocal laser scanning microscopy, Raman microspectroscopy, and atomic force microscopy. We hypothesize that these prosthecae allow for increased nutrient uptake by greatly expanding the cell surface area, providing a competitive advantage under nutrient-limited conditions.

Characterization of the First "Candidatus Nitrotoga" Isolate Reveals Metabolic Versatility and Separate Evolution of Widespread Nitrite-Oxidizing Bacteria.

Nitrification is a key process of the biogeochemical nitrogen cycle and of biological wastewater treatment. The second step, nitrite oxidation to nitrate, is catalyzed by phylogenetically diverse, chemolithoautotrophic nitrite-oxidizing bacteria (NOB). Uncultured NOB from the genus "Candidatus Nitrotoga" are widespread in natural and engineered ecosystems. Knowledge about their biology is sparse, because no genomic information and no pure "Ca Nitrotoga" culture was available. Here we obtained the first "Ca Nitrotoga" isolate from activated sludge. This organism, "Candidatus Nitrotoga fabula," prefers higher temperatures (>20°C; optimum, 24 to 28°C) than previous "Ca Nitrotoga" enrichments, which were described as cold-adapted NOB. "Ca Nitrotoga fabula" also showed an unusually high tolerance to nitrite (activity at 30 mM NO2-) and nitrate (up to 25 mM NO3-). Nitrite oxidation followed Michaelis-Menten kinetics, with an apparent Km (Km(app)) of ~89 µM nitrite and a Vmax of ~28 µmol of nitrite per mg of protein per h. Key metabolic pathways of "Ca Nitrotoga fabula" were reconstructed from the closed genome. "Ca Nitrotoga fabula" possesses a new type of periplasmic nitrite oxidoreductase belonging to a lineage of mostly uncharacterized proteins. This novel enzyme indicates (i) separate evolution of nitrite oxidation in "Ca Nitrotoga" and other NOB, (ii) the possible existence of phylogenetically diverse, unrecognized NOB, and (iii) together with new metagenomic data, the potential existence of nitrite-oxidizing archaea. For carbon fixation, "Ca Nitrotoga fabula" uses the Calvin-Benson-Bassham cycle. It also carries genes encoding complete pathways for hydrogen and sulfite oxidation, suggesting that alternative energy metabolisms enable "Ca Nitrotoga fabula" to survive nitrite depletion and colonize new niches.IMPORTANCE Nitrite-oxidizing bacteria (NOB) are major players in the biogeochemical nitrogen cycle and critical for wastewater treatment. However, most NOB remain uncultured, and their biology is poorly understood. Here, we obtained the first isolate from the environmentally widespread NOB genus "Candidatus Nitrotoga" and performed a detailed physiological and genomic characterization of this organism ("Candidatus Nitrotoga fabula"). Differences between key phenotypic properties of "Ca Nitrotoga fabula" and those of previously enriched "Ca Nitrotoga" members reveal an unexpectedly broad range of physiological adaptations in this genus. Moreover, genes encoding components of energy metabolisms outside nitrification suggest that "Ca Nitrotoga" are ecologically more flexible than previously anticipated. The identification of a novel nitrite-oxidizing enzyme in "Ca Nitrotoga fabula" expands our picture of the evolutionary history of nitrification and might lead to discoveries of novel nitrite oxidizers. Altogether, this study provides urgently needed insights into the biology of understudied but environmentally and biotechnologically important microorganisms.

MiDAS 2.0: an ecosystem-specific taxonomy and online database for the organisms of wastewater treatment systems expanded for anaerobic digester groups.

Wastewater is increasingly viewed as a resource, with anaerobic digester technology being routinely implemented for biogas production. Characterising the microbial communities involved in wastewater treatment facilities and their anaerobic digesters is considered key to their optimal design and operation. Amplicon sequencing of the 16S rRNA gene allows high-throughput monitoring of these systems. The MiDAS field guide is a public resource providing amplicon sequencing protocols and an ecosystem-specific taxonomic database optimized for use with wastewater treatment facility samples. The curated taxonomy endeavours to provide a genus-level-classification for abundant phylotypes and the online field guide links this identity to published information regarding their ecology, function and distribution. This article describes the expansion of the database resources to cover the organisms of the anaerobic digester systems fed primary sludge and surplus activated sludge. The updated database includes descriptions of the abundant genus-level-taxa in influent wastewater, activated sludge and anaerobic digesters. Abundance information is also included to allow assessment of the role of emigration in the ecology of each phylotype. MiDAS is intended as a collaborative resource for the progression of research into the ecology of wastewater treatment, by providing a public repository for knowledge that is accessible to all interested in these biotechnologically important systems. Database URL: http://www.midasfieldguide.org.

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.

Genomic and in situ investigations of the novel uncultured Chloroflexi associated with 0092 morphotype filamentous bulking in activated sludge.

Overgrowth of filamentous bacteria in activated sludge wastewater treatment plants (WWTPs) leads to impaired sludge settleability, a condition known as bulking, which is a common operational problem worldwide. Filaments with the Eikelboom 0092 morphotype are commonly associated with such bulking episodes. Members of the uncultured B45 phylotype, which is embraced within the phylum Chloroflexi, were recently shown to exhibit this morphology. Although these organisms are among the most abundant populations recorded in activated sludge processes, nothing is known about their metabolic characteristics. In this study, a genome sequence, representing the B45 phylotype, was retrieved from a metagenome generated from an activated sludge WWTP. The genome consisted of two chromosomes and one plasmid, which were 4.0, 1.0 and 0.04 Mbps in size, respectively. A metabolic model was constructed for this organism, based on annotation of its genome, showing its ability to generate energy by respiration, utilizing oxygen, nitrite or nitrous oxide as electron acceptors, or by fermentation of sugars. The ability of B45 members to ferment sugars under anaerobic conditions was validated in situ with microautoradiography-fluorescence in situ hybridization. The provisional name of 'Candidatus Promineofilum breve' is proposed for this species. This study represents the first detailed information on an uncultured genus of filamentous organisms from activated sludge.

MiDAS: the field guide to the microbes of activated sludge.

The Microbial Database for Activated Sludge (MiDAS) field guide is a freely available online resource linking the identity of abundant and process critical microorganisms in activated sludge wastewater treatment systems to available data related to their functional importance. Phenotypic properties of some of these genera are described, but most are known only from sequence data. The MiDAS taxonomy is a manual curation of the SILVA taxonomy that proposes a name for all genus-level taxa observed to be abundant by large-scale 16 S rRNA gene amplicon sequencing of full-scale activated sludge communities. The taxonomy can be used to classify unknown sequences, and the online MiDAS field guide links the identity to the available information about their morphology, diversity, physiology and distribution. The use of a common taxonomy across the field will provide a solid foundation for the study of microbial ecology of the activated sludge process and related treatment processes. The online MiDAS field guide is a collaborative workspace intended to facilitate a better understanding of the ecology of activated sludge and related treatment processes--knowledge that will be an invaluable resource for the optimal design and operation of these systems.

Metabolic model for the filamentous 'Candidatus Microthrix parvicella' based on genomic and metagenomic analyses.

'Candidatus Microthrix parvicella' is a lipid-accumulating, filamentous bacterium so far found only in activated sludge wastewater treatment plants, where it is a common causative agent of sludge separation problems. Despite attracting considerable interest, its detailed physiology is still unclear. In this study, the genome of the RN1 strain was sequenced and annotated, which facilitated the construction of a theoretical metabolic model based on available in situ and axenic experimental data. This model proposes that under anaerobic conditions, this organism accumulates preferentially long-chain fatty acids as triacylglycerols. Utilisation of trehalose and/or polyphosphate stores or partial oxidation of long-chain fatty acids may supply the energy required for anaerobic lipid uptake and storage. Comparing the genome sequence of this isolate with metagenomes from two full-scale wastewater treatment plants with enhanced biological phosphorus removal reveals high similarity, with few metabolic differences between the axenic and the dominant community 'Ca. M. parvicella' strains. Hence, the metabolic model presented in this paper could be considered generally applicable to strains in full-scale treatment systems. The genomic information obtained here will provide the basis for future research into in situ gene expression and regulation. Such information will give substantial insight into the ecophysiology of this unusual and biotechnologically important filamentous bacterium.