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Membrane-Aerated Biofilm Reactors (MABRs) are becoming a popular process intensification alternative within wastewater treatment plants (WWTP). Indeed, the nitrogen removal capacity of aerobic/anoxic/anaerobic reactors can be substantially enhanced with reduced energy consumption and footprint requirements. However, little is known about how oxidation–reduction potential (ORP) may impact their overall process performance. This study aims to report some of these effects by showing the results of almost three years of monitoring of two hybrid MABRs (R1, R2) adjacent to an existing Biodenipho™ facility. In Period 1 (P1), R1 and R2 were fed with anaerobic mixed liquor from the selector for the biological phosphorus removal zone. In Period 2 (P2), external aeration was introduced to increase ORP values (R1, R2), and membranes were replaced (R1) or cleaned (R2). Results show an increase in nitrification rates: from 0.27 and 0.33 g N m−2 d−1 in R1/R2 during P1 to 1.0 and 0.80 g N m−2 d−1 in R1/R2 during P2. 16 s rRNA amplicon sequencing analysis revealed that the relative abundance of nitrifying organisms increased from 0.2 to 6.7 % in R1 and 0.8 to 5.3 % in R2 in P2 (in detriment of microbes with fermenting capabilities). Energy dispersive X-ray spectroscopy confirmed the presence of coating substances under the lowest ORP (P1), which could be pyrite and its precursors like mackinawite. Overall, it is hypothesized that low ORP conditions (P1) had a detrimental effect on nitrification performance, as it promoted the reduction of different iron and sulfur compounds, which in turn a) precipitate in the biofilm as FeS increasing mass transfer limitations and competing with biomass for space; b) re-oxidize increasing the internal oxygen demand; c) inhibit nitrifiers growth.

Members of the genus Dechloromonas are often abundant in enhanced biological phosphorus removal (EBPR) systems and are recognized putative polyphosphate accumulating organisms (PAOs), but their role in phosphate removal is still unclear. Here, we used 16S rRNA gene sequencing and fluorescence in situ hybridization (FISH) to investigate the abundance and distribution of Dechloromonas spp. in Danish and global wastewater treatment plants. The two most abundant species worldwide revealed in situ dynamics of important intracellular storage polymers, measured by FISH-Raman in activated sludge from four full-scale EBPR plants and from a lab-scale reactor fed with different substrates. Moreover, seven distinct Dechloromonas species were determined from a set of ten high-quality metagenome-assembled genomes (MAGs) from Danish EBPR plants, each encoding the potential for polyphosphate (poly-P), glycogen, and polyhydroxyalkanoates (PHA) accumulation. The two species exhibited an in situ phenotype in complete accordance with the metabolic information retrieved by the MAGs, with dynamic levels of poly-P, glycogen, and PHA during feast-famine anaerobic–aerobic cycling, legitimately placing these microorganisms among the important PAOs. They are potentially involved in denitrification showing niche partitioning within the genus and with other important PAOs. As no isolates are available for the two species, we propose the names Candidatus Dechloromonas phosphoritropha and Candidatus Dechloromonas phosphorivorans.

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.

Large amounts of phosphorus (P) are present in activated sludge from municipal wastewater treatment plants, where it exists in the form of metal salt precipitates or biologically bound into the biomass as nucleic acids, cell membrane components, and the extracellular polymeric substances or, in special polyphosphate-accumulating organisms (PAOs), as intracellular polyphosphate. Only recently, methods that reliably allow an absolute quantification of the different P-fractions, such as sequential extraction, Raman microspectroscopy, solid-state 31P magic angle spinning (MAS) NMR, and solution state 31P NMR have been developed. This study combines these techniques to obtain a comprehensive P mass-balance of activated sludge from four wastewater treatment plants with enhanced biological phosphate removal (EBPR). The total content of P and various cations was measured by chemical analysis (ICP-OES), and different P fractions were extracted for chemical characterization. Chemically bound P constituted 38-69% of total P, most likely in the form of Fe, Mg, or Al minerals, while organically bound P constituted 7-9%. By using Raman microspectroscopy and solution state 31P NMR and 31P MAS NMR spectroscopy before and after anaerobic P-release experiments, poly-P was quantified and constituted 22-54% of total P in the activated sludges and was found in approx. 25% of all bacterial cells. Moreover, Raman microspectroscopy in combination with fluorescence in situ hybridization (FISH) was used to quantify the species-specific intracellular poly-P of known PAO genera (Tetrasphaera, Ca. Accumulibacter, Dechloromonas) and other microorganisms known to possess high level of poly-P, such as the filamentous Ca. Microthrix. They were all abundant, as measured by quantitative-FISH and amplicon sequencing, and accumulated large amount of poly-P, depending on their cell-size, contributing substantially to the P-removal. Interestingly, in all four EBPR plants investigated, only 1-13% of total poly-P was stored by unidentified PAO, highlighting that most PAOs in the full-scale EBPR plants investigated are now known.HighlightsExhaustive P mass-balance of main organic and inorganic P-species in four EBPR plantsQuantification of poly-P of FISH-defined PAO and other species with high P contentTotal P content was 36-50 mgP/gSS of which 31-62% was in biomass and as poly-PA high fraction of all cells (25-30%) contained a high content of poly-PKnown PAOs contained almost all poly-P in the EBPR plants investigated

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.

Phosphate (P) is a vital resource, but it is limited in its natural occurrence so recycling will become important to support the increasing demand. By using the Enhanced Biological Phosphorus Removal (EBPR) process in wastewater treatment plants (WWTPs), recycling of P from the wastewater is one way to combat this challenge. The EBPR process is controlled by polyphosphate-accumulating organisms (PAOs), such as the Candidatus Accumulibacter, Tetrasphaera, and Dechloromonas genera, which all store P intracellularly, despite of diverse metabolisms, depending on alternating anaerobic and aerobic conditions. New mass balance studies of biomass from full-scale plants with EBPR indicate that several unknown PAO exists, which may be important for the removal of P.The aim was to investigate the ecophysiology of Rhodoferax, Tessaracoccus, and Sulfuritalea, to determine whether they are PAOs or not. We used 16S rRNA gene sequencing to find the abundance, metagenomics to retrieve high-quality metagenome assembled genomes (MAGs) for annotation of metabolic pathways, and quantitative FISH-Raman analysis of polyphosphate (polyP) and other intracellular storage polymers to verify hypotheses. Rhodoferax and Sulfuritalea were observed in both Danish and global plants with up to 5.8% and 2.3% of total biomass, respectively. Tessaracoccus was found to contribute up to 2.6% in Danish plants. FISH-Raman analysis of Tessaracoccus and Rhodoferax showed signs of polyP. The chance of these two being PAOs was further supported by the retrieved MAGs, which showed the present of Pit, PstSCAB, and PhoU.By elucidating the ecophysiology of the remaining PAOs that contribute to phosphorus removal, we can enable surveillance and better control of the plants.

The activated sludge wastewater treatment process has been thoroughly researched in more than 100 years, yet there are still operational challenges that have not been fully resolved. Such a challenge is the control of filamentous bulking caused by the overgrowth of certain filamentous bacteria. In this study, we tested different mitigation strategies to reduce filamentous bulking, caused by two common filamentous genera found in full-scale water resource recovery facilities (WRRF), Candidatus Microthrix and Candidatus Amarolinea. PAX dosing, ozone addition, hydrocyclone implementation, and the addition of nano-coagulants were tested as mitigation strategies in four parallel treatment lines in a full-scale WRRF over three consecutive years. Unexpectedly, the activated sludge settleability was not affected by any of the mitigation strategies. Some of the strategies appeared to have a strong mitigating effect on the two filamentous species. However, detailed analyses of the microbial communities revealed strong recurrent seasonal variations in all four lines, including the control line which masked the real effect. After removing the effect of the seasonal variation by using a time-series decomposition approach, it was clear that the filamentous bacteria were mostly unaffected by the mitigation strategies. Only PAX dosing had some effect on Ca. Microthrix, but only on one species, Ca. Microthrix subdominans, and not on the most common Ca. Microthrix parvicella. Overall, our study shows the importance of long-term monitoring of microbial communities at species level to understand the normal seasonal pattern to effectively plan and execute full-scale experiments. Moreover, the results highlight the importance of using parallel reference treatment lines when evaluating the effect of mitigation strategies in full-scale treatment plants.

We have critically assessed some of the dogmas in the microbiology of enhanced biological phosphorus removal (EBPR) and argue that the genus Tetrasphaera can be as important as Ca. Accumulibacter for phosphorus removal; and that proliferation of their competitors, the glycogen accumulating organisms, does not appear to be a practical problem for EBPR efficiency even under tropical conditions. An increasing number of EBPR-related genomes are changing our understanding of their physiology, for example, their potential to participate in denitrification. Rather than trying to identify organisms that adhere to strict phenotype metabolic models, we advocate for broader analyses of the whole microbial communities in EBPR plants by iterative studies with isolates, lab enrichments, and full-scale systems.

Significance Wastewater treatment plants are engineering technologies used worldwide to protect the environment and human health. Microbial communities sustain these plants, so it is crucial to know the key factors responsible for the community assembly. We show, in contrast to existing understanding, that microbial immigration largely controls the community structure in these plants and that the fate (growth or death) of immigrating species in the plants is controlled by local factors. The community structure was quantitatively predicted by the immigrating microbial community, highlighting the need to revise the way we today understand, design, and manage microbial communities in wastewater treatment plants., 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.

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.

Anaerobic digestion (AD) is a key technology at many wastewater treatment plants (WWTPs) for converting primary and surplus activated sludge to methane-rich biogas. However, the limited number of surveys and the lack of comprehensive datasets have hindered a deeper understanding of the characteristics and associations between key variables and the microbial community composition. Here, we present a six-year survey of 46 anaerobic digesters, located at 22 WWTPs in Denmark, which is the first and largest known study of the microbial ecology of AD at WWTPs at a regional scale. For three types of AD (mesophilic, mesophilic with thermal hydrolysis pretreatment, and thermophilic), we present the typical value range of 12 key parameters including operational variables and performance parameters. High-resolution bacterial and archaeal community analyses were carried out at species level using amplicon sequencing of >1,000 samples and the new ecosystem-specific MiDAS 3 reference database. We detected 42 phyla, 1,600 genera, and 3,584 species in the bacterial community, where 70% of the genera and 93% of the species represented environmental taxa that were only classified based on MiDAS 3 de novo placeholder taxonomy. More than 40% of the bacterial species were found not to grow in the mesophilic and thermophilic digesters and were only present due to immigration with the feed sludge. Ammonium concentration was the main driver shaping the bacterial community while temperature and pH were main drivers for the archaea in the three types of ADs. Sub-setting for the growing microbes improved significantly the correlation analyses and revealed the main drivers for the presence of specific species. Within mesophilic digesters, feed sludge composition and other key parameters (organic loading rate, biogas yield, and ammonium concentration) correlated with specific growing species. This survey provides a comprehensive insight into community structure at species level, providing a foundation for future studies of the ecological significance/characteristics and function of the many novel or poorly described taxa.

The function of the microbiomes in wastewater treatment systems and anaerobic digesters is dictated by the physiological activity of their members and complex interactions between them. Since functional traits are often conserved at low taxonomic ranks (genus, species, strain), high resolution taxonomic classification is crucial to understand the role of microbes in any ecosystem. Here we present MiDAS 3, a comprehensive 16S rRNA gene reference database based on full-length 16S rRNA gene amplicon sequence variants (FL-ASVs) derived from activated sludge and anaerobic digester systems in Denmark. The new database proposes unique provisional names for all unclassified microorganisms down to species level, providing a new and much-needed tool for microbiome research. The MiDAS 3 database was used to analyze the microbiome in 20 Danish wastewater treatment plants with nutrient removal, sampled over 13 years. The 50 most abundant species belonged to 42 genera, including 14 genera with provisional ‘midas’ name. Of those, 20 have no known function in the system, which highlights the need for more efforts towards elucidating the role of important members of wastewater treatment ecosystems. The new MiDAS 3 database also forms the backbone of the MiDAS Field Guide – an online resource linking the identity of microorganisms in wastewater treatment systems to available data related to their functional importance. The new field guide contains a complete list of genera (>1800) and species (>4200) found in activated sludge and anaerobic digesters in Denmark, but is also relevant to wastewater systems across the world. The identity of the microbes is linked to functional information, where available, and the website provides the possibility to BLAST new sequences against the MiDAS 3 database. The MiDAS Field Guide is a collaborative platform acting as an online knowledge repository, facilitating understanding of wastewater treatment ecosystem function.

Anaerobic digestion (AD) is a key technology at many wastewater treatment plants (WWTPs) for converting surplus activated sludge to methane-rich biogas. However, the limited number of surveys and the lack of comprehensive data sets have hindered a deeper understanding of the characteristics and associations between key variables and the microbiome composition. Here, we present a six-year survey of 46 anaerobic digesters, located at 22 WWTPs in Denmark, which is the largest known study of the microbial ecology of AD at WWTPs at a regional scale. For three types of AD (mesophilic, mesophilic with thermal hydrolysis pretreatment, and thermophilic), we present the typical value range of 12 key parameters including operational variables and performance parameters. The bacterial and archaeal microbiomes were analyzed at species-level resolution using amplicon sequencing in >1,000 samples and the new ecosystem-specific MiDAS 3 reference database. We detected 42 phyla, 1,600 genera and 3,584 species in the bacterial microbiome, where 70% of the genera and 93% of the species represented uncultivated taxa that were only classified based on MiDAS 3 denovo placeholder taxonomy. More than 40% of the 100 most abundant bacterial species did not grow in the digesters and were only present due to immigration with the feed sludge. Temperature, ammonium concentration, and pH were the main drivers shaping the microbiome clusters of the three types of ADs for both bacteria and for archaea. Within mesophilic digesters, feed sludge composition and other key parameters (organic loading rate, biogas yield, and ammonium concentration) correlated with the growing bacterial microbiome. Furthermore, correlation analysis revealed the main drivers for specific species among growing bacteria and archaea, and revealed the potential ecological function of many novel taxa. Our study highlights the influence of immigration on bacterial AD microbiome. Subsetting the growing microbes improves the understanding of the diversity and main drivers of microbiome assembly, and elucidates functionality of specific species-level microorganisms. This six-year survey provides a comprehensive insight into microbiome structure at species level, engineering and ecological performance, and a foundation for future studies of the ecological significance/characteristics and function of the novel taxa.

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.

Membrane bioreactors (MBRs) for wastewater treatment show great potentials in the sustainable development of urban environments. However, fouling of membranes remains the largest challenge of MBR technology. Dissolved extracellular polymeric substances (EPS) are often assumed be the main foulant in MBRs. However, single bacterial cells are often erroneously measured as EPS in traditional spectrophotometric analysis of EPS in activated sludge, so we hypothesized that single cells in many cases could be the true foulants in MBRs for wastewater treatment. To study this, raw MBR sludge and sludge supernatant with varying concentrations of planktonic cells were filtered on microfiltration (MF) membranes, and we found a direct correlation between the cell count and rate of flux decline. Addition of planktonic cells to fresh MBR sludge dramatically increased the flux decline. The identity of the most abundant planktonic cells in a full-scale MBR water resource recovery facility was determined by DNA fingerprinting. Many of these genera are known to be abundant in influent wastewater suggesting that the influent bacterial cells may have a direct effect on the fouling propensity in MBR systems. This new knowledge may lead to new anti-fouling strategies targeting incoming planktonic bacteria from the wastewater feed. Practitioner points: Planktonic cells constituted up to 60% of the total protein content of “soluble extracellular polymeric substances” in membrane bioreactor sludge. Planktonic cells are hidden under a surrogate concentration of extracellular polymeric substances which is often associated with fouling. Membrane fouling rate is directly proportional to amount of free planktonic cells suspended in sludge. Several influent bacterial genera are enriched in the water phase of membrane bioreactor sludge. Removing these may mitigate fouling.

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.

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.

Members of the genus Dechloromonas are often abundant in enhanced biological phosphorus removal (EBPR) systems and are recognized putative polyphosphate accumulating organisms (PAOs), but their role in phosphate (P) removal is still unclear. Here, we used 16S rRNA gene sequencing and fluorescence in situ hybridization (FISH) to investigate the abundance and distribution of Dechloromonas spp. in Danish wastewater treatment plants. Two species were abundant, novel, and uncultured, and could be targeted by existing FISH probes. Raman microspectroscopy of probe-defined organisms (FISH-Raman) revealed the levels and dynamics of important intracellular storage polymers in abundant Dechloromonas spp. in the activated sludge from four full-scale EBPR plants and from a lab-scale sequencing batch reactor fed with different carbon sources (acetate, glucose, glycine, and glutamate). Moreover, 7 distinct Dechloromonas species were determined from a set of 10 high-quality metagenome-assembled genomes (MAGs) from Danish EBPR plants, each encoding the potential for poly-P, glycogen, and polyhydroxyalkanoates (PHA) accumulation. The two most abundant species exhibited an in situ phenotype in complete accordance with the metabolic information retrieved by the MAGs, with dynamic levels of poly-P, glycogen, and PHA during feast-famine anaerobic-aerobic cycling, legitimately placing these microorganisms among the important PAOs. As no isolates are available for the two species, we propose the names Candidatus Dechloromonas phosphatis and Candidatus Dechloromonas phosphovora.

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.

Recent development of sequencing techniques has enabled us to create new ecosystem-specific full-length 16S rRNA gene reference databases, which gives a unique opportunity to design FISH probes for novel bacteria and revise existing FISH-probes. By using ecosystem-specific instead of universal large-scale databases, it is possible to design specific FISH probes for nearly all microbes in the given ecosystem, allowing visualization of novel microbial key players and combine these for studies of their ecophysiology. Here we have used a wastewater treatment-specific database to design FISH-probes for novel members of the phylum Acidobacteria, a hitherto overlooked but abundant group in activated sludge. Combination with Raman microspectroscopy provided functional information

Members of the genus Dechloromonas are often abundant in EBPR plants worldwide and have long been considered putative PAOs, as intracellular poly-P has been identified with traditional staining methods. This study applied metagenomics to determine the metabolic potential and Raman microspectroscopy to verify and define the levels and dynamics of important storage polymers in FISH-defined Dechloromonas cells. A 99.2% complete metagenome-assembled genome, retrieved from Danish EBPR plants, showed the potential for poly-P, glycogen, and PHA accumulation. Dechloromonas cells exhibited in situ a Ca. Accumulibacter-like phenotype with dynamic levels of poly-P and PHA during feast-famine cycling. Quantitative Raman-FISH analysis of single cells from full-scale activated sludge plants confirmed Dechloromonas to be an important PAO.

The function of microbial communities in wastewater treatment systems and anaerobic digesters is dictated by the physiological activity of its members and complex interactions between them. Since functional traits are often conserved at low taxonomic ranks (genus, species, strain), the development of high taxonomic resolution and reliable classification is the first crucial step towards understanding the role of microbes in any ecosystem. Here we present MiDAS 3, a comprehensive 16S rRNA gene reference database based on high-quality full-length sequences derived from activated sludge and anaerobic digester systems. The MiDAS 3 taxonomy proposes unique provisional names for all microorganisms down to species level. MiDAS 3 was applied for the detailed analysis of microbial communities in 20 Danish wastewater treatment plants with nutrient removal, sampled over 12 years, demonstrating community stability and many abundant core taxa. The top 50 most abundant species belonged to genera, of which >50% have no known function in the system, emphasizing the need for more efforts towards elucidating the role of important members of wastewater treatment ecosystems. The MiDAS 3 taxonomic database guided an update of the MiDAS Field Guide – an online resource linking the identity of microorganisms in wastewater treatment systems to available data related to their functional importance. The new field guide contains a complete list of genera (>1,800) and species (>4,200) found in activated sludge and anaerobic digesters. The identity of the microbes is linked to functional information, where available. The website also provides the possibility to BLAST the sequences against MiDAS 3 taxonomy directly online. The MiDAS Field Guide is a collaborative platform acting as an online knowledge repository and facilitating understanding of wastewater treatment ecosystem function.

Comprehensive surveys from full-scale wastewater treatment plants consistently show high abundance of potential polyphosphate accumulating organisms (PAO) in full-scale plants besides the well-studied Candidatus Accumulibacter. However, the diversity, ecophysiology and functionality of these organisms remain largely unresolved. Targeted laboratory-scale experiments combined with monitoring the microbial evolution and microbial performance showed a diverse community with several novel PAO candidates. By using the new ecosystem-specific reference database MiDAS 3 for high-resolution amplicon analyses and design of novel highly specific FISH probes, several novel species within the genus Tetrasphaera and other genera were revealed as potential PAOs. By combining cycle studies with Raman-FISH for ecophysiological studies, the relative contribution of the different novel PAOs to P-removal under different conditions can be resolved.

Dissolved extracellular polymeric substances (EPS) are often mentioned as the main foulant in membrane bioreactors (MBR). However, single bacterial cells may be the most important foulant but are often erroneously measured as EPS in traditional spectrophotometric analysis. Raw sludge and sludge supernatant with varying concentrations of planktonic cells was filtered, and showed a direct correlation between the planktonic cell count and rate of flux decline. Thus, high concentrations of single cells result in membrane fouling. DNA fingerprinting of activated sludge samples from a full scale MBR wastewater treatment plants (WWTP) was applied to determine the identity of the most abundant single cells. Several bacteria genera were observed to be enriched in the water phase, many of them known to be abundant in influent wastewater and these may influence fouling propensity in MBR systems.

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) 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. The AutoTax taxonomy greatly improves the classification of short-read 16S rRNA gene amplicon sequence variants (ASVs) at the genus- and species-level, compared to 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.

Enhanced biological phosphorus removal (EBPR) is a globally important biotechnological process and relies on the massive accumulation of phosphate within special microorganisms. Candidatus Accumulibacter conform to the classical physiology model for polyphosphate accumulating organisms and are widely believed to be the most important player for the process in full-scale EBPR systems. However, it was impossible till now to quantify the contribution of specific microbial clades to EBPR. In this study, we have developed a new tool to directly link the identity of microbial cells to the absolute quantification of intracellular poly-P and other polymers under in situ conditions, and applied it to eight full-scale EBPR plants. Besides Ca. Accumulibacter, members of the genus Tetrasphaera were found to be important microbes for P accumulation, and in six plants they were the most important. As these Tetrasphaera cells did not exhibit the classical phenotype of poly-P accumulating microbes, our entire understanding of the microbiology of the EBPR process has to be revised. Furthermore, our new single-cell approach can now also be applied to quantify storage polymer dynamics in individual populations in situ in other ecosystems and might become a valuable tool for many environmental microbiologists.

Filamentous bacteria belonging to the phylum Chloroflexi have received considerable attention in wastewater treatment systems for their suggested role in the operational problem of impaired sludge settleability known as bulking. Their consistently high abundance in full-scale systems, even in the absence of bulking, indicates that they make a substantial contribution to the nutrient transformations during wastewater treatment. In this study, extensive 16S rRNA amplicon surveys of Danish wastewater treatment plants (WWTPs) with nutrient removal were screened to identify numerically important Chloroflexi genera. Fluorescence in situ hybridization probes were designed for their in situ characterization. All abundant Chloroflexi phylotypes were putatively identified as facultative anaerobic chemoorganotrophs involved in sugar fermentation. They were all filamentous but differed in their morphology and spatial arrangement. 'Candidatus Villigracilis' was predominantly located within the activated sludge flocs, where they possibly have structural importance, and their abundance was relatively stable. Conversely, the abundance of 'Candidatus Amarolinea' was highly dynamic, relative to other genera, sometimes reaching abundances in excess of 30% of the biovolume, suggesting their likely role in bulking episodes. This study gives an important insight into the role of Chloroflexi in WWTPs, thus contributing to the broader goal of understanding the ecology of these biotechnologically important systems.

Cable bacteria of the family Desulfobulbaceae form centimeter-long filaments comprising thousands of cells. They occur worldwide in the surface of aquatic sediments, where they connect sulfide oxidation with oxygen or nitrate reduction via long-distance electron transport. In the absence of pure cultures, we used single-filament genomics and metagenomics to retrieve draft genomes of 3 marine Candidatus Electrothrix and 1 freshwater Ca. Electronema species. These genomes contain >50% unknown genes but still share their core genomic makeup with sulfate-reducing and sulfur-disproportionating Desulfobulbaceae, with few core genes lost and 212 unique genes (from 197 gene families) conserved among cable bacteria. Last common ancestor analysis indicates gene divergence and lateral gene transfer as equally important origins of these unique genes. With support from metaproteomics of a Ca. Electronema enrichment, the genomes suggest that cable bacteria oxidize sulfide by reversing the canonical sulfate reduction pathway and fix CO 2 using the Wood–Ljungdahl pathway. Cable bacteria show limited organotrophic potential, may assimilate smaller organic acids and alcohols, fix N 2 , and synthesize polyphosphates and polyglucose as storage compounds; several of these traits were confirmed by cell-level experimental analyses. We propose a model for electron flow from sulfide to oxygen that involves periplasmic cytochromes, yet-unidentified conductive periplasmic fibers, and periplasmic oxygen reduction. This model proposes that an active cable bacterium gains energy in the anodic, sulfide-oxidizing cells, whereas cells in the oxic zone flare off electrons through intense cathodic oxygen respiration without energy conservation; this peculiar form of multicellularity seems unparalleled in the microbial world.

Full scale wastewater treatment plants are usually considered to perform well in general removal of bacteria coming in with sewage. Therefore it is assumed that bacteria highly abundant in the process tanks and effluent comprise primarily of those that have active growth in the plant. However standard methods for detecting pathogenic bacteria in effluent are highly based on culture dependent methods, which may give erroneous results by underestimating the bacteria that are not removed. The aim of this study was to determine the fate of immigrating pathogenic bacteria with a specific focus on an underestimated pathogen group the genus Arcobacter. And investigate with culture independent methods if these are discharged with the effluent. Culture independent 16S rRNA gene amplicon sequencing was applied for the identification of bacteria. In influent, process tanks and effluent at 14 Danish full–scale WWTPs. Net growth rates and cell numbers were calculated for specific OTU’s using bacterial mass balances. Amplicon studies showed that the genus Arcobacter was abundant in all 14 investigated plants. Mass balances confirmed that Arcobacter did not grow in the plants indicating that cells of Arcobacter originated from the influent. The pathogenic species Arcobacter butzleri was isolated. Identity and presence of virulence genes were confirmed by genomics studies. Results indicates that specific genera stay in the water phase and are directly discharged undetected by the current culture-based methods. Other procedures and methods need to be implemented to successfully identify and remove bacteria like Arcobacter before they are discharged with the effluent.

The activated sludge process is key in modern wastewater treatment. The microbial community composition must be stable in order to maintain the desired nutrient removal. In addition, bacterial morphology, mode of growth and extracellular polymeric substances composition determine floc size, shape and strength, which in turn influence important sludge properties. Optimal sludge properties are important for the wastewater treatment plant operation and eventually effluent quality. The aim of this study was to established the link between the microbial community structure and physico-chemical sludge characteristics and thereby provide a better understanding of the activated sludge process.DNA-based identification of microorganisms using 16S rRNA gene amplicon sequencing has been proved as a reliable and reproducible method for identification and quantification studies of microbial communities in activated sludge and for the demonstration of their significance for plant operation and stability. 16S rRNA gene amplicon sequencing has been developed over the past few years and is now ready to use for more comprehensive studies related to plant operation and optimization thanks to short analysis time, low cost, high throughput, and high taxonomic resolution.In this study, we found that the composition of microbial community differs between different sludge fractions and that specific bacteria are enriched in defloculated fraction (supernatant) and in the fraction loosely bound to the floc. In addition, individual bacterial types were found to correlate with the defined floc properties.

Enhanced biological phosphorus removal (EBPR) is a globally important biotechnological process and relies on the massive accumulation of phosphate within special microorganisms.CandidatusAccumulibacter conform to classical physiology model for polyphosphate accumulating organisms and are widely believed to be the most important player for the process in full-scale EBPR systems. However, it was impossible till now to quantify the contribution of specific microbial clades to EBPR. In this study, we have developed a new tool to directly link the identity of microbial cells to the absolute quantification of intracellular poly-P and other polymers underin situconditions, and applied it to eight full-scale EBPR plants. BesidesCa. Accumulibacter, members of the genusTetrasphaerawere found to be important microbes for P accumulation, and in six plants they were the most important. As theseTetrasphaeracells did not exhibit the classical phenotype of poly-P accumulating microbes, our entire understanding of the microbiology of the EBPR process has to be revised. Furthermore, our new single-cell approach can now also be applied to quantify storage polymer dynamics in individual populationsin situin other ecosystems and might become a valuable tool for many environmental microbiologists.

Filamentous bacteria belonging to the phylum Chloroflexi have received considerable attention in wastewater treatment systems for their suggested role in operational problem of impaired sludge settleability known as bulking. Their consistently high abundance in full-scale systems, even in the absence of bulking, indicates that they make a substantial contribution to the nutrient transformations during wastewater treatment. In this study, extensive 16S rRNA amplicon surveys of full-scale Danish WWTPs were screened to identify the most numerically important Chloroflexi genera. Fluorescencein situhybridization probes were designed for theirin situcharacterization. All abundant phylotypes of the phylum were identified as facultative anaerobic chemoorganotrophs involved in fermentation of sugars. These groups were all filamentous but differed in their morphology and spatial arrangement.‘CandidatusVilligracilis’ was predominantly located within the activated sludge flocs, where they possibly have structural importance, and their abundance was relatively stable. Conversely, the abundance of‘CandidatusAmarolinea’ was highly dynamic, relative to other genera, sometimes reaching abundances in excess of 30% of the biovolume, suggesting their likely role in bulking episodes. This study gives an important insight into the role of Chloroflexi in WWTPs, thus contributing to the broader goal of understanding the ecology of these biotechnologically important systems.