Fluorescently probing anaerobic digester sludge: Measuring single-cell anabolic activity in methanogens (Methanosarcina and Methanothermobacter) with deuterium-labeled Raman analysis.

This study addresses the challenge of obtaining in situ information on substrate utilization rates for individual microbial species in complex microbial communities such as anaerobic digester sludge. To overcome this hurdle, a novel approach combining doubly-labelled deuterium, fluorescence in situ hybridization (FISH) and Raman microspectroscopy was developed. The method enables quantitative determination of anabolic heavy hydrogen incorporation into FISH-targeted, exemplified by methanogenic cells from the genera Methanosarcina and Methanothermobacter. The deuterium incorporation rates ascertained by Raman red-shifting of C-Hx vibrational region to C-Dx vibrations, quantified through Raman peak area ratios, were compared for different carbon sources. Methanosarcina exhibited highest kinetic rates with acetate and propionate, while Methanothermobacter demonstrated faster incorporation under acetate and methanol supplementation. This groundbreaking study demonstrates the feasibility of obtaining quantitative metabolic rate information at a single-cell level using deuterium, FISH probes, and Raman microspectroscopy.

The predicted secreted proteome of activated sludge microorganisms indicates distinct nutrient niches.

In wastewater treatment plants (WWTPs), complex microbial communities process diverse chemical compounds from sewage. Secreted proteins are critical because many are the first to interact with or degrade external (macro)molecules. To better understand microbial functions in WWTPs, we predicted secreted proteomes of WWTP microbiota from more than 1,000 high-quality metagenome-assembled genomes (MAGs) from 23 Danish WWTPs with biological nutrient removal. Focus was placed on examining secreted catabolic exoenzymes that target major classes of macromolecules. We demonstrate that Bacteroidota has a high potential to digest complex polysaccharides, but also proteins and nucleic acids. Poorly understood activated sludge members of Acidobacteriota and Gemmatimonadota also have high capacities for extracellular polysaccharide digestion. Secreted nucleases are encoded by 61% of MAGs indicating an importance for extracellular DNA and/or RNA digestion in WWTPs. Secreted lipases were the least common macromolecule-targeting enzymes predicted, encoded mainly by Gammaproteobacteria and Myxococcota. In contrast, diverse taxa encode extracellular peptidases, indicating that proteins are widely used nutrients. Diverse secreted multi-heme cytochromes suggest capabilities for extracellular electron transfer by various taxa, including some Bacteroidota that encode undescribed cytochromes with >100 heme-binding motifs. Myxococcota have exceptionally large secreted protein complements, probably related to predatory lifestyles and/or complex cell cycles. Many Gammaproteobacteria MAGs (mostly former Betaproteobacteria) encode few or no secreted hydrolases, but many periplasmic substrate-binding proteins and ABC- and TRAP-transporters, suggesting they are mostly sustained by small molecules. Together, this study provides a comprehensive overview of how WWTPs microorganisms interact with the environment, providing new insights into their functioning and niche partitioning.IMPORTANCEWastewater treatment plants (WWTPs) are critical biotechnological systems that clean wastewater, allowing the water to reenter the environment and limit eutrophication and pollution. They are also increasingly important for the recovery of resources. They function primarily by the activity of microorganisms, which act as a "living sponge," taking up and transforming nutrients, organic material, and pollutants. Despite much research, many microorganisms in WWTPs are uncultivated and poorly characterized, limiting our understanding of their functioning. Here, we analyzed a large collection of high-quality metagenome-assembled genomes from WWTPs for encoded secreted enzymes and proteins, with special emphasis on those used to degrade organic material. This analysis showed highly distinct secreted proteome profiles among different major phylogenetic groups of microorganisms, thereby providing new insights into how different groups function and co-exist in activated sludge. This knowledge will contribute to a better understanding of how to efficiently manage and exploit WWTP microbiomes.

Microbial core communities in activated sludge plants are strongly affected by immigration and geography.

BACKGROUND: The microbiota in wastewater treatment plants (WWTPs) and incoming wastewater is critical for the treatment process, the preservation of natural ecosystems and human health, and for the recovery of resources and achievement of sustainability goals. Both core species and conditionally rare and abundant taxa (CRAT) are considered process-critical but little is known about identity as well as true functional and ecological importance. Here, we present a comprehensive investigation of the microbiota of 84 municipal activated sludge (AS) plants with nutrient removal treating ~ 70% of all wastewater within a confined geographical area, Denmark (43,000 km2). With the use of an ecosystem-specific database (MiDAS 5.2), species-level classification allowed us to investigate the core and CRAT species, whether they were active, and important factors determining their presence. RESULTS: We established a comprehensive catalog of species with names or placeholder names showing each plant contained approx. 2,500 different species. Core and CRAT represented in total 258 species, constituting around 50% of all reads in every plant. However, not all core and CRAT could be regarded as process-critical as growth rate calculations revealed that 43% did not grow in the AS plants and were present only because of continuous immigration from the influent. Analyses of regional microbiota differences and distance decay patterns revealed a stronger effect for species than genera, demonstrating that geography had a clear effect on the AS microbiota, even across a limited geographical area such as Denmark (43,000 km2). CONCLUSIONS: The study is the first comprehensive investigation of WWTPs in a confined geographical area providing new insights in our understanding of activated sludge microbiology by introducing a concept of combining immigration and growth calculation with identifying core and CRAT to reveal the true ecosystem-critical organisms. Additionally, the clear biogeographical pattern on this scale highlights the need for more region-level studies to find regional process-critical taxa (core and CRAT), especially at species and amplicon sequence variant (ASV) level.

MiDAS 5: Global diversity of bacteria and archaea in anaerobic digesters.

Anaerobic digestion of organic waste into methane and carbon dioxide (biogas) is carried out by complex microbial communities. Here, we use full-length 16S rRNA gene sequencing of 285 full-scale anaerobic digesters (ADs) to expand our knowledge about diversity and function of the bacteria and archaea in ADs worldwide. The sequences are processed into full-length 16S rRNA amplicon sequence variants (FL-ASVs) and are used to expand the MiDAS 4 database for bacteria and archaea in wastewater treatment systems, creating MiDAS 5. The expansion of the MiDAS database increases the coverage for bacteria and archaea in ADs worldwide, leading to improved genus- and species-level classification. Using MiDAS 5, we carry out an amplicon-based, global-scale microbial community profiling of the sampled ADs using three common sets of primers targeting different regions of the 16S rRNA gene in bacteria and/or archaea. We reveal how environmental conditions and biogeography shape the AD microbiota. We also identify core and conditionally rare or abundant taxa, encompassing 692 genera and 1013 species. These represent 84-99% and 18-61% of the accumulated read abundance, respectively, across samples depending on the amplicon primers used. Finally, we examine the global diversity of functional groups with known importance for the anaerobic digestion process.

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.

Candidate biomarkers of antibiotic resistance for the monitoring of wastewater and the downstream environment.

Urban wastewater treatment plants (UWTPs) are essential for reducing the pollutants load and protecting water bodies. However, wastewater catchment areas and UWTPs emit continuously antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs), with recognized impacts on the downstream environments. Recently, the European Commission recommended to monitor antibiotic resistance in UWTPs serving more than 100 000 population equivalents. Antibiotic resistance monitoring in environmental samples can be challenging. The expected complexity of these systems can jeopardize the interpretation capacity regarding, for instance, wastewater treatment efficiency, impacts of environmental contamination, or risks due to human exposure. Simplified monitoring frameworks will be essential for the successful implementation of analytical procedures, data analysis, and data sharing. This study aimed to test a set of biomarkers representative of ARG contamination, selected based on their frequent human association and, simultaneously, rare presence in pristine environments. In addition to the 16S rRNA gene, ten potential biomarkers (intI1, sul1, ermB, ermF, aph(3'')-Ib, qacEΔ1, uidA, mefC, tetX, and crAssphage) were monitored in DNA extracts (n = 116) from raw wastewater, activated sludge, treated wastewater, and surface water (upstream and downstream of UWTPs) samples collected in the Czech Republic, Denmark, Israel, the Netherlands, and Portugal. Each biomarker was sensitive enough to measure decreases (on average by up to 2.5 log-units gene copy/mL) from raw wastewater to surface water, with variations in the same order of magnitude as for the 16S rRNA gene. The use of the 10 biomarkers allowed the typing of water samples whose origin or quality could be predicted in a blind test. The results show that, based on appropriate biomarkers, qPCR can be used for a cost-effective and technically accessible approach to monitoring wastewater and the downstream environment.

Improved description of terrestrial habitat types by including microbial communities as indicators.

Soils host diverse communities of microorganisms essential for ecosystem functions and soil health. Despite their importance, microorganisms are not covered by legislation protecting biodiversity or habitats, such as the Habitats Directive. Advances in molecular methods have caused breakthroughs in microbial community analysis, and recent studies have shown that parts of the communities are habitat-specific. If distinct microbial communities are present in the habitat types defined in the Habitats Directive, the Directive may be improved by including these communities. Thus, monitoring and reporting of biodiversity and conservation status of habitat types could be based not only on plant communities but also on microbial communities. In the present study, bacterial and plant communities were examined in six habitat types defined in the Habitats Directive by conducting botanical surveys and collecting soil samples for amplicon sequencing across 19 sites in Denmark. Furthermore, selected physico-chemical properties expected to differ between habitat types and explain variations in community composition of bacteria and vegetation were analysed (pH, electrical conductivity (EC), soil texture, soil water repellency, soil organic carbon content (OC), inorganic nitrogen, and in-situ water content (SWC)). Despite some variations within the same habitat type and overlaps between habitat types, habitat-specific communities were observed for both bacterial and plant communities, but no correlation was observed between the alpha diversity of vegetation and bacteria. PERMANOVA analysis was used to evaluate the variables best able to explain variation in the community composition of vegetation and bacteria. Habitat type alone could explain 46% and 47% of the variation in bacterial and plant communities, respectively. Excluding habitat type as a variable, the best model (pH, SWC, OC, fine silt, and Shannon's diversity index for vegetation) could explain 37% of the variation for bacteria. For vegetation, the best model (pH, EC, ammonium content and Shannon's diversity index for bacteria) could explain 25% of the variation. Based on these results, bacterial communities could be included in the Habitats Directive to improve the monitoring, as microorganisms are more sensitive to changes in the environment compared to vegetation, which the current monitoring is based on.

Primary settling changes the microbial community of influent wastewater to wastewater treatment plants.

The continuous immigration of bacteria in influent wastewater strongly impacts the microbial community of activated sludge (AS) in wastewater treatment plants (WWTP), both in terms of species composition and their abundance. Therefore, it is of interest to elucidate the route of immigrating bacteria into the biological tanks, including the effect of primary settlers. These are commonly used pretreatment units that can possibly selectively increase or reduce the relative abundance of certain bacteria. Species-level identification of the microbial composition of influent wastewater before and after primary settling was carried out in four full-scale municipal WWTPs biweekly over one year by 16S rRNA gene amplicon sequencing. Overall, 37-49% of incoming COD was removed in the primary settlers. Most genera and species were present in the wastewater to all four plants and the trend of these were investigated across the primary settlers. Approximately 50% of the genera had the same trend across at least three WWTPs. Few genera significantly increased in relative read abundance (3.7%) after settling, while 22.3% showed a significant reduction in relative abundance. We investigated process-critical species in AS, such as known nitrifiers, polyphosphate-accumulating organisms, and filamentous bacteria. Most taxa were affected similarly in all WWTPs including multiple genera involved in bulking in AS. However, some genera, e.g., important polyphosphate-accumulating bacteria, had inconsistent trends across WWTPs, suggesting that the characteristics of the wastewater are important for the trend of some bacteria through primary settling. In all cases, primary settling changed the microbial community of the influent wastewater, posing an obvious candidate for upstream control to optimize the assembly of the microbial communities in activated sludge.

Toward a Universal Unit for Quantification of Antibiotic Resistance Genes in Environmental Samples.

Surveillance of antibiotic resistance genes (ARGs) has been increasingly conducted in environmental sectors to complement the surveys in human and animal sectors under the "One-Health" framework. However, there are substantial challenges in comparing and synthesizing the results of multiple studies that employ different test methods and approaches in bioinformatic analysis. In this article, we consider the commonly used quantification units (ARG copy per cell, ARG copy per genome, ARG density, ARG copy per 16S rRNA gene, RPKM, coverage, PPM, etc.) for profiling ARGs and suggest a universal unit (ARG copy per cell) for reporting such biological measurements of samples and improving the comparability of different surveillance efforts.

Fast DNA-analyses for surveillance of microbial communities in full-scale deammonification tanks: Potential for control and troubleshooting.

The partial nitritation/anammox process is a popular process for sidestream nitrogen removal, but the process is sensitive to disturbances and requires extensive surveillance and monitoring for optimal performance. We followed two parallel sidestream full-scale deammonification reactors treating digester centrate for a year with high time-resolution of both online sensor data and microbial community as measured by Nanopore DNA sequencing. DNA surveillance revealed system disturbances and allowed for detection of process and equipment upsets, and it facilitated remediating operational actions. Surveillance of anammox bacteria (Ca. Brocadia) revealed unexpected variations, and the composition and dynamics of the flanking community indicated causes for occasional process disturbances with poor nitrogen removal. Monitoring the ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) could potentially allow reactor operation with increased dissolved oxygen (DO), yielding higher ammonia conversion while keeping NOB in control. The use of fast and frequent DNA sequencing (sampling 3-5 times a week, analysed once per week) was an important supplement, and in many cases superior, to the online sensor data for process surveillance, understanding and control.

Closed genomes uncover a saltwater species of Candidatus Electronema and shed new light on the boundary between marine and freshwater cable bacteria.

Cable bacteria of the Desulfobulbaceae family are centimeter-long filamentous bacteria, which are capable of conducting long-distance electron transfer. Currently, all cable bacteria are classified into two candidate genera: Candidatus Electronema, typically found in freshwater environments, and Candidatus Electrothrix, typically found in saltwater environments. This taxonomic framework is based on both 16S rRNA gene sequences and metagenome-assembled genome (MAG) phylogenies. However, most of the currently available MAGs are highly fragmented, incomplete, and thus likely miss key genes essential for deciphering the physiology of cable bacteria. Also, a closed, circular genome of cable bacteria has not been published yet. To address this, we performed Nanopore long-read and Illumina short-read shotgun sequencing of selected environmental samples and a single-strain enrichment of Ca. Electronema aureum. We recovered multiple cable bacteria MAGs, including two circular and one single-contig. Phylogenomic analysis, also confirmed by 16S rRNA gene-based phylogeny, classified one circular MAG and the single-contig MAG as novel species of cable bacteria, which we propose to name Ca. Electronema halotolerans and Ca. Electrothrix laxa, respectively. The Ca. Electronema halotolerans, despite belonging to the previously recognized freshwater genus of cable bacteria, was retrieved from brackish-water sediment. Metabolic predictions showed several adaptations to a high salinity environment, similar to the "saltwater" Ca. Electrothrix species, indicating how Ca. Electronema halotolerans may be the evolutionary link between marine and freshwater cable bacteria lineages.

Full-scale activated sludge transplantation reveals a highly resilient community structure.

Well-functioning and stable microbial communities are critical for the operation of activated sludge (AS) wastewater treatment plants (WWTPs). Bioaugmentation represents a potentially useful approach to recover deteriorated systems or to support specific AS processes, but its application in full-scale WWTPs is generally problematic. We conducted a massive transplantation (in one day) exchanging AS from a donor to a recipient full-scale WWTP with similar process type (biological removal of nitrogen and phosphorus) and performance, but with differences in microbial community structure. The treatment performance in the recipient plant was not compromised and the effluent quality remained stable. The AS community structure of the recipient plant was initially very similar to the donor AS, but it almost completely restored the pre-transplantation structure approximately 40 days after transplantation, corresponding to 3 times the solid retention time. Most of the unique species of donor AS added to recipient AS disappeared quickly, although some disappeared more slowly the following months, indicating some survival and potentially a time limited function in the recipient plant. Moreover, the addition in higher abundance of most species already present in the recipient AS (e.g., the polyphosphate accumulating organisms) or the reduction of the abundance of unwanted bacteria (e.g., filamentous bacteria) in the recipient plant was not successful. Moreover, we observed similar abundance patterns after transplantation for species belonging to different functional guilds, so we did not observe an increase of the functional redundancy. Investigations of the microbial community structure in influent wastewater revealed that for some species the abundance trends in the recipient plant were closely correlated to their abundance in the influent. We showed that a very resilient microbial community was responsible for the outcome of the transplantation of AS at full-scale WWTP, potentially as a consequence of mass-immigration from influent wastewater. The overall results imply that massive transplantation of AS across different WWTPs is not a promising strategy to permanently solve operational problems. However, by choosing a compatible AS donor, short term mitigation of serious operational problems may be possible.

Genetic potential for exopolysaccharide synthesis in activated sludge bacteria uncovered by genome-resolved metagenomics.

A good floc formation of activated sludge (AS) is crucial for solid-liquid separation and production of clean effluent during wastewater treatment. Floc formation is partly controlled by self-produced extracellular polymeric substances (EPS) such as exopolysaccharides, proteins, and nucleic acids. Little is known about the composition, structure, and function of EPS in AS and which bacteria produce them. To address this knowledge gap for the exopolysaccharides, we took advantage of 1083 high-quality metagenome-assembled genomes (MAGs) obtained from 23 Danish wastewater treatment plants. We investigated the genomic potential for exopolysaccharide biosynthesis in bacterial species typical in AS systems based on genome mining and gene synteny analyses. Putative gene clusters associated with the biosynthesis of alginate, cellulose, curdlan, diutan, hyaluronic acids, Pel, poly-ß-1,6-N-acetyl-d-glucosamine (PNAG), Psl, S88 capsular polysaccharide, salecan, succinoglycan, and xanthan were identified and linked to individual MAGs, providing a comprehensive overview of the genome-resolved potential for these exopolysaccharides in AS bacteria. The approach and results provide a starting point for a more comprehensive understanding of EPS composition in wastewater treatment systems, which may facilitate a more refined regulation of the activated sludge process for improved stability.

Comparative analysis of floc characteristics and microbial communities in anoxic and aerobic suspended growth processes.

A fully anoxic suspended growth process is an appealing alternative to conventional activated sludge (AS) due to considerable aeration reduction and improved carbon processing efficiency for biological nutrient removal (BNR). With development of the hybrid membrane aerated biofilm reactor (MABR) technology, implementation of a fully anoxic suspended growth community in BNR facilities became practical. To better understand potential limitations with the elimination of aeration, we carried out microscopic examination and 16S rRNA gene-based microbial community profiling to determine how an anoxic suspended growth would differ from the conventional aerobic process in floc characteristics, microbial diversity, microbial temporal dynamics, and community assembly pattern. Fewer filamentous populations were found in the anoxic mixed liquor, suggesting easily sheared flocs. The anoxic microbial community had distinct composition and structure, but its diversity and temporal dynamics were similar to the conventional aerobic community. A variety of well-studied functional guilds were also identified in the anoxic community. The anoxic microbial community assembly was more stochastic than the conventional aerobic community, but deterministic assembly was still significant with a large core microbiome adapted to the anoxic condition. PRACTITIONER POINTS: Flocs developed under the anoxic conditions had less filamentous backbones, implying reduced flocculation capacity and easily sheared flocs. Knowledge about the ecophysiology of Thauera, Thiothrix, and Trichococcus can help achieve good properties of the anoxic flocs. A diverse microbial community sustainably adapted to the fully anoxic condition, containing a variety of filaments, denitrifiers, and PAOs. The anoxic microbial community displayed a similar degree of diversity and temporal dynamics compared to the aerobic counterpart. The anoxic community's assembly was more stochastic, so it may be less subject to changes in environmental variables.

Exploring the microbial influence on seasonal nitrous oxide concentration in a full-scale wastewater treatment plant using metagenome assembled genomes.

Nitrous oxide is a highly potent greenhouse gas and one of the main contributors to the greenhouse gas footprint of wastewater treatment plants (WWTP). Although nitrous oxide can be produced by abiotic reactions in these systems, biological N2O production resulting from the imbalance of nitrous oxide production and reduction by microbial populations is the dominant cause. The microbial populations responsible for the imbalance have not been clearly identified, yet they are likely responsible for strong seasonal nitrous oxide patterns. Here, we examined the seasonal nitrous oxide concentration pattern in Avedøre WWTP alongside abiotic parameters, the microbial community composition based on 16S rRNA gene sequencing and already available metagenome-assembled genomes (MAGs). We found that the WWTP parameters could not explain the observed pattern. While no distinct community changes between periods of high and low dissolved nitrous oxide concentrations were determined, we found 26 and 28 species with positive and negative correlations to the seasonal N2O concentrations, respectively. MAGs were identified for 124 species (approximately 31% mean relative abundance of the community), and analysis of their genomic nitrogen transformation potential could explain this correlation for four of the negatively correlated species. Other abundant species were also analysed for their nitrogen transformation potential. Interestingly, only one full-denitrifier (Candidatus Dechloromonas phosphorivorans) was identified. 59 species had a nosZ gene predicted, with the majority identified as a clade II nosZ gene, mainly from the phylum Bacteroidota. A correlation of MAG-derived functional guilds with the N2O concentration pattern showed that there was a small but significant negative correlation with nitrite oxidizing bacteria and species with a nosZ gene (N2O reducers (DEN)). More research is required, specifically long-term activity measurements in relation to the N2O concentration to increase the resolution of these findings.

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.

Quantification of Biologically and Chemically Bound Phosphorus in Activated Sludge from Full-Scale Plants with Biological P-Removal.

Phosphorus (P) is present in activated sludge from wastewater treatment plants in the form of metal salt precipitates, extracellular polymeric substances, or bound into the biomass, for example, as intracellular polyphosphate (poly-P). Several methods for a reliable quantification of the different P-fractions have recently been developed, and this study combines them to obtain a comprehensive P mass-balance of activated sludge from four enhanced biological phosphate removal (EBPR) plants. Chemical characterization by ICP-OES and sequential P fractionation showed that chemically bound P constituted 38-69% of total P, most likely in the form of Fe, Mg, or Al minerals. Raman microspectroscopy, solution state 31P NMR, and 31P MAS NMR spectroscopy applied before and after anaerobic P-release experiments, were used to quantify poly-P, which constituted 22-54% of total P and was found in approximately 25% of all bacterial cells. Raman microspectroscopy in combination with fluorescence in situ hybridization was used to quantify poly-P in known polyphosphate-accumulating organisms (PAO) (Tetrasphaera, Candidatus Accumulibacter, and Dechloromonas) and other microorganisms known to possess high level of poly-P, such as the filamentous Ca. Microthrix. Interestingly, only 1-13% of total P was stored by unidentified PAO, highlighting that most PAOs in the full-scale EBPR plants investigated are known.

Microbial communities across activated sludge plants show recurring species-level seasonal patterns.

Microbial communities in activated sludge (AS) are the core of sanitation in wastewater treatment plants (WWTPs). Microbial communities in AS have shown seasonal changes, however, long-term experiments (>2 years) are rarely conducted, limiting our understanding of the true seasonal dynamics in WWTPs. In this study, we resolved the microbial seasonal dynamics at the species level in four municipal full-scale WWTPs, sampled every 7-10 days, during 3-5 consecutive years. By applying a new time-series analysis approach, we revealed that the seasonal pattern was species-specific, where species belonging to the same functional guild or genus may show different seasonal dynamics. Species could be grouped into cohorts according to their seasonal patterns, where seasonal cohorts showed repeatable annual dynamics across years and plants. Species were also grouped according to their net growth rate in the AS (i.e., growing species and disappearing species). Growing species were more prevailing in spring and autumn cohorts, while disappearing species, which were only present due to the continuous immigration from influent wastewater, were mostly associated with winter and spring cohorts. Most known process-critical species, such as nitrifiers, polyphosphate accumulating organisms and filamentous organisms, showed distinct species-specific patterns. Overall, our study showed that overarching seasonal patterns affected microbial species in full-scale AS plants, with similar seasonal patterns across plants for many dominant species. These recurrent seasonal variations should be taken into account in the operation, understanding and management of the WWTPs.