Mass-immigration determines the assembly of activated sludge microbial communities.

The assembly of bacterial communities in wastewater treatment plants (WWTPs) is affected by immigration via wastewater streams, but the impact and extent of bacterial immigrants are still unknown. Here, we quantify the effect of immigration at the species level in 11 Danish full-scale activated sludge (AS) plants. All plants have different source communities but have very similar process design, defining the same overall environmental growth conditions. The AS community composition in each plant was strongly reflected by the corresponding influent wastewater (IWW) microbial composition. Most species in AS across the plants were detected and quantified in the corresponding IWW, allowing us to identify their fate in the AS: growing, disappearing, or surviving. Most of the abundant species in IWW disappeared in AS, so their presence in the AS biomass was only due to continuous mass-immigration. In AS, most of the abundant growing species were present in the IWW at very low abundances. We predicted the AS species abundances from their abundance in IWW by using a partial least square regression model. Some species in AS were predicted by their own abundance in IWW, while others by multiple species abundances. Detailed analyses of functional guilds revealed different prediction patterns for different species. We show, in contrast to the present understanding, that the AS microbial communities were strongly controlled by the IWW source community and could be quantitatively predicted by taking into account immigration. This highlights a need to revise the way we understand, design, and manage the microbial communities in WWTPs.

Bacteria from the Genus Arcobacter Are Abundant in Effluent from Wastewater Treatment Plants.

Pathogenic bacteria in wastewater are generally considered to be efficiently removed in biological wastewater treatment plants. This understanding is almost solely based on culture-based control measures, and here we show, by applying culture-independent methods, that the removal of species in the genus Arcobacter was less effective than for many other abundant genera in the influent wastewater. Arcobacter was one of the most abundant genera in influent wastewater at 14 municipal wastewater treatment plants and was also abundant in the "clean" effluent from all the plants, reaching up to 30% of all bacteria as analyzed by 16S rRNA gene amplicon sequencing. Metagenomic analyses, culturing, genome sequencing of Arcobacter isolates, and visualization by fluorescent in situ hybridization (FISH) confirmed the presence of the human-pathogenic Arcobacter cryaerophilus and A. butzleri in both influent and effluent. The main reason for the high relative abundance in the effluent was probably that Arcobacter cells, compared to those of other abundant genera in the influent, did not flocculate and attach well to the activated sludge flocs, leaving a relatively large fraction dispersed in the water phase. The study shows there is an urgent need for new standardized culture-independent measurements of pathogens in effluent wastewaters, e.g., amplicon sequencing, and an investigation of the problem on a global scale to quantify the risk for humans and livestock.IMPORTANCE The genus Arcobacter was unexpectedly abundant in the effluent from 14 Danish wastewater treatment plants treating municipal wastewater, and the species included the human-pathogenic A. cryaerophilus and A. butzleri Recent studies have shown that Arcobacter is common in wastewater worldwide, so the study indicates that discharge of members of the genus Arcobacter may be a global problem, and further studies are needed to quantify the risk and potentially minimize the discharge. The study also shows that culture-based analyses are insufficient for proper effluent quality control, and new standardized culture-independent measurements of effluent quality encompassing most pathogens should be considered.

Global abundance patterns, diversity, and ecology of Patescibacteria in wastewater treatment plants.

BACKGROUND: Microorganisms are responsible for nutrient removal and resource recovery in wastewater treatment plants (WWTPs), and their diversity is often studied by 16S rRNA gene amplicon sequencing. However, this approach underestimates the abundance and diversity of Patescibacteria due to the low coverage of commonly used PCR primers for this highly divergent bacterial phylum. Therefore, our current understanding of the global diversity, distribution, and ecological role of Patescibacteria in WWTPs is very incomplete. This is particularly relevant as Patescibacteria are considered to be associated with microbial host cells and can therefore influence the abundance and temporal variability of other microbial groups that are important for WWTP functioning. RESULTS: Here, we evaluated the in silico coverage of widely used 16S rRNA gene-targeted primer pairs and redesigned a primer pair targeting the V4 region of bacterial and archaeal 16S rRNA genes to expand its coverage for Patescibacteria. We then experimentally evaluated and compared the performance of the original and modified V4-targeted primers on 565 WWTP samples from the MiDAS global sample collection. Using the modified primer pair, the percentage of ASVs classified as Patescibacteria increased from 5.9 to 23.8%, and the number of detected patescibacterial genera increased from 560 to 1576, while the detected diversity of the remaining microbial community remained similar. Due to this significantly improved coverage of Patescibacteria, we identified 23 core genera of Patescibacteria in WWTPs and described the global distribution pattern of these unusual microbes in these systems. Finally, correlation network analysis revealed potential host organisms that might be associated with Patescibacteria in WWTPs. Interestingly, strong indications were found for an association between Patescibacteria of the Saccharimonadia and globally abundant polyphosphate-accumulating organisms of the genus Ca. Phosphoribacter. CONCLUSIONS: Our study (i) provides an improved 16S rRNA gene V4 region-targeted amplicon primer pair inclusive of Patescibacteria with little impact on the detection of other taxa, (ii) reveals the diversity and distribution patterns of Patescibacteria in WWTPs on a global scale, and (iii) provides new insights into the ecological role and potential hosts of Patescibacteria in WWTPs. Video Abstract.

The Parkinson's drug entacapone disrupts gut microbiome homeostasis via iron sequestration.

Increasing evidence shows that many human-targeted drugs alter the gut microbiome, leading to implications for host health. However, much less is known about the mechanisms by which drugs target the microbiome and how drugs affect microbial function. Here we combined quantitative microbiome profiling, long-read metagenomics, stable isotope probing and single-cell chemical imaging to investigate the impact of two widely prescribed nervous system-targeted drugs on the gut microbiome. Ex vivo supplementation of physiologically relevant concentrations of entacapone or loxapine succinate to faecal samples significantly impacted the abundance of up to one third of the microbial species present. Importantly, we demonstrate that the impact of these drugs on microbial metabolism is much more pronounced than their impact on abundances, with low concentrations of drugs reducing the activity, but not the abundance of key microbiome members like Bacteroides, Ruminococcus or Clostridium species. We further demonstrate that entacapone impacts the microbiome due to its ability to complex and deplete available iron, and that microbial growth can be rescued by replenishing levels of microbiota-accessible iron. Remarkably, entacapone-induced iron starvation selected for iron-scavenging organisms carrying antimicrobial resistance and virulence genes. Collectively, our study unveils the impact of two under-investigated drugs on whole microbiomes and identifies metal sequestration as a mechanism of drug-induced microbiome disturbance.

High Diversity and Functional Potential of Undescribed "Acidobacteriota" in Danish Wastewater Treatment Plants.

Microbial communities in water resource recovery facilities encompass a large diversity of poorly characterized lineages that could have undescribed process-critical functions. Recently, it was shown that taxa belonging to "Acidobacteriota" are abundant in Danish full-scale activated sludge wastewater treatment plants (WWTP), and here we investigated their diversity, distribution, and functional potential. "Acidobacteriota" taxa were identified using a comprehensive full-length 16S rRNA gene reference dataset and amplicon sequencing surveys across 37 WWTPs. Members of this phylum were diverse, belonging to 14 families, eight of which are completely uncharacterized and lack type strains. Several lineages were abundant, with relative abundances of up to 5% of the microbial community. Genome annotation and metabolic reconstruction of 50 high-quality "Acidobacteriota" metagenome-assembled genomes (MAGs) from 19 WWTPs showed high metabolic diversity and potential involvement in nitrogen and phosphorus removal and iron reduction. Fluorescence in situ hybridization (FISH) using newly-designed probes revealed cells with diverse morphologies, predominantly located inside activated sludge flocs. FISH in combination with Raman microspectroscopy revealed ecophysiological traits in probe-defined cells from the families Holophagaceae, Thermoanaerobaculaceae, and Vicinamibacteraceae, and families with the placeholder name of midas_f_502, midas_f_973, and midas_f_1548. Members of these lineages had the potential to be polyphosphate-accumulating organisms (PAOs) as intracellular storage was observed for the key compounds polyphosphate and glycogen.

A refined set of rRNA-targeted oligonucleotide probes for in situ detection and quantification of ammonia-oxidizing bacteria.

Ammonia-oxidizing bacteria (AOB) of the betaproteobacterial genera Nitrosomonas and Nitrosospira are key nitrifying microorganisms in many natural and engineered ecosystems. Since many AOB remain uncultured, fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes has been one of the most widely used approaches to study the community composition, abundance, and other features of AOB directly in environmental samples. However, the established and widely used AOB-specific 16S rRNA-targeted FISH probes were designed up to two decades ago, based on much smaller rRNA gene sequence datasets than available today. Several of these probes cover their target AOB lineages incompletely and suffer from a weak target specificity, which causes cross-hybridization of probes that should detect different AOB lineages. Here, a set of new highly specific 16S rRNA-targeted oligonucleotide probes was developed and experimentally evaluated that complements the existing probes and enables the specific detection and differentiation of the known, major phylogenetic clusters of betaproteobacterial AOB. The new probes were successfully applied to visualize and quantify AOB in activated sludge and biofilm samples from seven pilot- and full-scale wastewater treatment systems. Based on its improved target group coverage and specificity, the refined probe set will facilitate future in situ analyses of AOB.

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.

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.