Water Research

A novel integrated pilot-scale A-stage high rate activated sludge, B-stage short-cut biological nitrogen removal and side-stream enhanced biological phosphorus removal (A/B-shortcut N- S2EBPR) process for treating municipal wastewater was demonstrated with the aim to achieve simultaneous and carbon- and energy-efficient N and P removal. In this studied period, an average of 7.62 {+/-} 2.17 mg-N/L nitrite accumulation was achieved through atypical partial nitrification without canonical known NOB out-selection. Network analysis confirms the central hub of microbial community as Nitrospira, which was one to two orders of magnitude higher than canonical aerobic oxidizing bacteria (AOB) in a B-stage nitrification tank. The contribution of comammox Nitrospira as AOB was evidenced by the increased amoB/nxr ratio and higher ammonia oxidation activity. Furthermore, oligotyping analysis of Nitrospira revealed two dominant sub-clusters (microdiveristy) within the Nitrospira. The relative abundance of oligotype II, which is phylogenetically close to Nitrospira_midas_s_31566, exhibited a positive correlation with nitrite accumulation in the same operational period, suggesting its role as comammox Nitrospira. Additionally, the phylogenetic investigation suggested that heterotrophic organisms from the family Comamonadacea and the order Rhodocyclaceae embedding ammonia monooxygenase and hydroxylamine oxidase may function as heterotrophic nitrifiers. This is the first study that elucidated the impact of integrating the S2EBPR on nitrifying populations with implications on short-cut N removal. The unique conditions in the side-stream reactor, such as low ORP, favorable VFA concentrations and composition, seemed to exert different selective forces on nitrifying populations from those in conventional biological nutrient removal processes. The results provide new insights for integrating EBPR with short-cut N removal process for mainstream wastewater treatment.

Granular sludge intensifies the removal of nutrients from wastewater. Granules structured by extracellular polymeric substances (EPS) can be recovered as biomaterial. Links between microbial selection and EPS formation during granulation need to get uncovered. We inoculated anaerobic-aerobic sequencing batch reactors with either flocs or granules to study the relationships between microbial selection, bioaggregation, exopolymer formation, and EPS composition. Selection for slow-growing organisms like the model polyphosphate- accumulating organism "Candidatus Accumulibacter" (max. 83% vs. amplicon sequencing read counts) and glycogen-accumulating organism "Ca. Competibacter" (max. 45%) sustained granulation. Gel-forming exopolymers were produced as high as above 40% of the volatile solids of the biomass by stepwise increase of the organic loading rate (0.3 to 2.0 g CODAc d-1 LR-1). Confocal laser scanning microscopy, FT-IR spectroscopy, and HPAE-PAD chromatography revealed the complex and dynamic chemical compositions of the structural EPS in relation to microbial population shifts along reactor regimes. The analysis of 20 representative genomes of "Ca. Accumulibacter" and "Ca. Competibacter" recovered from public databases revealed their functional potential to produce EPS among other representative wastewater microorganisms. The more than 40 functional gene categories annotated highlight the complexity of EPS metabolic networks from monomers processing to assembly, export, and epimerizations. The combination of ecological engineering principles and systems microbiology will help unravel and direct the production of EPS from wastewater, valorizing residual granular sludge into beneficial biomaterials for the circular economy. HighlightsO_LISelection for slow-growing organisms like PAOs and GAOs fostered a robust granulation. C_LIO_LIStructural EPS were produced above 40% of biomass volatile content under high loading. C_LIO_LIChemical composition of EPS evolved together with the microbial community composition. C_LIO_LIGenomic insights highlighted the genetic potential of PAOs and GAOs for EPS formation. C_LIO_LIMicrobial communities are complex; further are their EPS compositions and metabolisms. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=130 SRC="FIGDIR/small/534144v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@a163e2org.highwire.dtl.DTLVardef@1a4ab94org.highwire.dtl.DTLVardef@1fc93d4org.highwire.dtl.DTLVardef@14d4b7f_HPS_FORMAT_FIGEXP M_FIG C_FIG

The presence of biofilms in drinking water distribution systems (DWDS) can affect both water quality and system integrity; yet these systems remain poorly studied due to lack of accessibility. We established two independent full-scale DWDS Testbeds (A and B) on two different campuses situated in a tropical urban environment and equipped them with online sensors. Testbed B experienced higher levels of monochloramine and lower water age than Testbed A within the campus. Based on long amplicon-sequencing of bacterial 16S rRNA genes extracted from the mature biofilms (MPB) growing on pipes and young biofilms (YSB) growing on the sensors, a core community was identified in the two testbeds. The relative abundances of operational taxonomic units at the family level, including Mycobacteriaceae, Methylobacteriaceae, Rhodospirillaceae, Nitrosomonadaceae, and Moraxellaceae, were consistent for MPB and YSB on each campus. The MPB community was found to be influenced by conductivity, sample age, and pipe diameter as determined by both canonical correlation analysis and fuzzy set ordination. MPB displayed higher -diversity based on Hill numbers than YSB; in general, second order Hill numbers correlated positively with conductivity and sample age, but negatively with ORP and nitrite. Pseudomonas spp. together with Bacillus spp. likely initiated biofilm formation of YSB on Testbed A under conditions of reduced monochloramine and high water age. Significant levels of orthophosphate were detected in YSB samples at two stations and associated with higher levels of stagnation based on long-term differential turbidity measurement (DTM). Orthophosphate and DTM may act as indicators of the biofilm growth potential within DWDS. Highlights- Established two testbeds to study biofilms in full-scale distribution system - Biofilms on pipes and sensors had core community - Temporal effect and higher -diversity for biofilms on pipes - Water chemistry was related to biofilm community differences O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/434709v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@c392aforg.highwire.dtl.DTLVardef@1d355eborg.highwire.dtl.DTLVardef@1addf37org.highwire.dtl.DTLVardef@19cc22_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

Side-stream Enhanced biological phosphorus removal (S2EBPR) has been incorporated with B-stage process to enable simultaneous phosphorus and nitrogen removal. However, the dominating phosphorus accumulating organisms (PAOs) in this novel configuration has not been evaluated. The dominance of Acinetobacter was confirmed by 16S sequencing. In addition, single cell Raman spectrum (SCRS) analysis in couple with in situ fluorescence in situ hybridization (FISH) was applied to obtain the feature spectrum and verify the phosphorus release/uptake activity of Acinetobacter spp. The phenotypic profiling further suggested the dominance of Acinetobacter-like organisms among all poly-phosphorus containing organisms and only certain phenotypic Acinetobacter (oligotype 1) contribution to P-removal in a unique HRAS-P(D)N-S2EBPR system. The findings suggest that Acinetobacter may outcompete other heterotrophic organisms in EBPR systems due to their sensitivity to operational conditions. However, stable P-removal was only observed during a specific section of the operation period, coinciding with an increase in the VFA/P ratio. Further research is needed to identify the phenotypes of Acinetobacter responsible for P-removal in EBPR systems. The study contributes to a better understanding of the microbial ecology and engineering aspects of EBPR systems and wastewater treatment in general.

Propionic acid (HPr) accumulation is a major indicator of anaerobic digestion (AD) dysfunction, yet the relative contributions of acidity, undissociated HPr, and propionate ions (Pr-) to process inhibition remain poorly understood. We investigated these effects in mesophilic batch AD microcosms fed with municipal sewage sludge, using a comparative design involving HPr, sodium propionate (NaPr), NaCl, and HCl treatments across two series of experiments. While 20 mM HPr caused a 22% reduction in the maximal methane production rate, 81 mM HPr led to complete inhibition, with the initial pH dropping to 5.1. By contrast, 81 mM NaPr reduced methane production rate by only 40%, and 81 mM NaCl caused no inhibition, demonstrating that acidity is the dominant inhibitory factor, with Pr- exerting a secondary concentration-dependent effect. 16S rRNA gene amplicon sequencing revealed strong, compound-specific shifts in microbial community composition, affecting key functional groups including syntrophs and methanogenic archaea. The proportion of methanogens dropped from 2-3% in control reactors to less than 0.2% under 81 mM HPr, consistent with the observed methane production inhibition. Under HPr81, over 100 ASVs were differentially abundant compared to controls, a pattern largely shared with HCl-treated reactors, further confirming the predominant role of acidity. The number of differentially abundant ASVs was negatively correlated with methane production rates (R{superscript 2} = 0.97), underscoring the link between community reshaping and process impairment. These results provide a unifying framework for propionate inhibition in AD and suggest that microbial community profiling could serve as an early warning tool for process imbalance detection.

Harmful cyanobacterial blooms present persistent risks to both drinking water security and wastewater reuse, driving the need for advanced treatment strategies. Treatment barrier(s) need to be capable of simultaneously controlling cyanobacteria, cyanotoxins, and co-occurring contaminants like bloom-associated antimicrobial resistance genes (ARGs) particularly in the case of recycling treated wastewater. Ozone nanobubble technology has emerged as a promising innovation, offering extended oxidative stability and enhanced interfacial reactivity compared to conventional ozonation. Hence this research objectives were to (a) assess the removal performance of ozone nanobubbles in eliminating cyanobacteria and their co-occurring contaminants in comparison to conventional ozone systems, and (b) investigate the repeatability of the results in varying background water qualities, ozone decay and the potential for by-products formation Ozonation using nanobubbles enhanced oxidation performance by 19%-34% in the drinking water reservoir compared to conventional ozonation while keeping the ozone concentration below 2mg/l. Lower oxidation efficiencies were observed in treated wastewater compared to drinking water sources, reflecting the higher content of organic matter and suspended solids, and oxidant demand characteristic of recycled water systems. Despite these challenges, ozone nanobubbles consistently outperformed conventional ozonation in reducing both cyanobacterial biomass and cell viability, underscoring their potential as an advanced "polishing" step for algal management in wastewater reuse applications. By exploring fate of ARGs alongside cyanobacteria and toxin removal, this work extends beyond traditional ozonation trials. It provides valuable field-based evidence that bridges the divide between laboratory efficacy and full-scale operational performance. Future studies should build on this by exploring combined or sequential treatment barriers that enhance DNA degradation, thereby addressing both cellular and genetic risks in water supply and reuse systems. Observing the action of nanobubbles under dynamic, real-world water quality conditions is currently challenging; however, this studys novel field trials demonstrate potential nanobubble applications and provide valuable insights to guide future investigations. The results reinforce the broader applicability of ozone nanobubble technology for multi-target contaminant control in water reservoirs.

River microbial communities regularly act as the first defense barrier against the spread of antimicrobial resistance genes (ARG) that enter environmental microbiomes through wastewaters. However, how the invasion dynamics of wastewater-born ARGs into river biofilm communities will shift due to increasing average and peak temperatures worldwide through climate change remains unknown. Here we aimed at elucidating the effects of increasing temperatures on both, the natural river biofilm resistome, as well as the river biofilms invadability by foreign, wastewater-born ARGs. To achieve this, natural biofilms were grown in a pristine German river and transferred to artificial laboratory recirculation flume systems at three different temperatures (20{degrees}C, 25{degrees}C, 30{degrees}C). Already after one week of acclimatization to the temperatures, significant increases in the abundance of most naturally occurring ARGs were detected in the biofilms exposed to the highest temperature. Thereafter, biofilms were exposed to a single pulse of wastewater and the invasion dynamics of wastewater-born ARGs were analyzed over a period of two weeks. While initially after one day ARGs were able to invade all biofilms successfully and in equal proportions, the foreign invading ARGs were lost at a far increased rate at 30{degrees}C over time. ARG levels dropped to the initial natural levels at 30{degrees}C after 14 days. Contrary at the lower temperatures ARGs remained far elevated and certain ARGs were able to establish themselves in the biofilms. Overall, we here demonstrate tradeoffs of increasing temperature between increases in naturally occurring and faster loss dynamics of invading ARGs.

Pharmaceutical residues are persistent contaminants that resist conventional wastewater treatment and can disrupt ecosystems; however, microorganisms provide a promising biobased solution to transform or mineralize these complex xenobiotics. Whether pollutant-adapted communities maintain their degradative capacity under realistic environmental conditions remains a long-standing debate in environmental biotechnology. Here, microbial consortia enriched in six membrane bioreactors under high pharmaceutical concentration (100 mg/L) retained full biodegradation capacity across a 5000-fold concentration range. After prolonged exposure to six model compounds (atenolol, caffeine, diclofenac, enalapril, ibuprofen, and paracetamol) complete removal occurred for all except diclofenac. Degradation remained efficient even at lower and environmentally relevant concentrations (1 mg/L-20 {micro}g/L) and recovered rapidly upon re-exposure to higher loads (100 mg/L). Metagenomic profiling revealed enrichment of oxygenase-mediated catabolic pathways supporting this resilience. When transferred to a 7 liters bioreactor treating real wastewater, the adapted community removed targeted and untargeted pharmaceuticals, demonstrating robustness, scalability, and strong potential for sustainable micropollutant remediation. Environmental ImplicationPharmaceuticals and their metabolites are environmentally hazardous because these bioactive micropollutants are persistent and continuously discharged via wastewater, thereby endangering both ecosystem and human health. This study shows that pollutant-adapted microbial consortia can address this challenge, retaining strong degradative function across large concentration fluctuations, including environmentally relevant levels. It also demonstrates scalability: the adapted community can be transferred to real-wastewater operation to remove both targeted and additional pharmaceuticals, supporting a bio-based "polishing" step for wastewater treatment plants. Overall, these findings support more sustainable biological mitigation strategies to reduce micropollutant loads.

Proteins are an abundant extracellular polymeric substance (EPS) of activated sludge from wastewater. Hot alkalinization is implemented industrially to recover the EPS from sludge. We sought to assess the feasibility of protein recovery from alkali EPS. We detected a low abundance of bacterial proteins in the EPS. Human keratin was highly abundant and could also be recovered. Keratin was observed as a dominant and integral component of the activated sludge flocs, and it was thus not an extraction artifact. Alkali extraction promoted Maillard reaction between proteins and sugars and removed recoverable peptide signatures. Subsequent chemical modification, along with denaturation, impaired protein binding to ion exchange resins, making bacterial proteins inaccessible to isolation. Keratin has high resistance to Maillard reaction under extraction conditions and thus persists in the EPS. While Maillard modifies bacterial proteins, the resultant product, and possibly even keratin itself, are valuable recoverable byproducts from activated sludge. SynopsisThe different sensitivities of proteins to Maillard reaction determines which proteins dominate alkaline extracellular polymeric substance (EPS) extract from activated sludge, with keratin dominating in alkaline EPS and an integral activated sludge component.

Noroviruses (NoV) are the leading cause of non-bacterial gastroenteritis across the globe with societal costs of US$60.3 billion per annum. Development of a long amplicon nanopore-based method for dual-typing the RNA-dependent RNA polymerase (RdRp) and major structural protein (VP1) regions from a single RNA fragment could improve existing norovirus typing methods. Its application to wastewater-based epidemiology (WBE) and environmental testing could enable the discovery of novel types and improve tracking throughout the population and into aquaculture and recreational water settings. Here, we develop and optimise such a method for wastewater as the sample matrix. Reverse transcription (RT), PCR and library pooling were optimised and a consensus-based bioinformatics pipeline was developed. Inhibitor removal and LunaScript(R) RT gave robust amplification of the {approx}1000 bp RdRP+VP1 amplicon. Platinum Taq polymerase showed good sensitivity and reduced levels non-specific amplification (NSA) when compared to other polymerases. Optimised PCR annealing temperatures significantly reduced NSA (51.3% and 42.4% for GI and GII), increased yield (86.5% for GII) and increased taxa richness (57.7%) for GII. Analysis of three NoV positive faecal samples showed 100% nucleotide similarity with Sanger sequencing. Eight GI genotypes, 11 polymerase types (p-types) and 13 combinations were detected in wastewater along with 4 GII genotypes, 4 p-types and 8 combinations; highlighting the diversity of norovirus taxa present in wastewater in England. The most common genotypes detected in clinical samples were all detected in wastewater while we also commonly detected several GI genotypes not reported in the clinical data. Application of this method into a WBE scheme, therefore, may allow for more accurate measurement of norovirus diversity within the population.

Antimicrobial resistance (AMR) is an ever-increasing threat to global health. Wastewater-based surveillance is an emerging methodology that objectively enables an inclusive and comprehensive assessment of population AMR in an observed sewershed. Here we compared the resistome of two tertiary-care hospitals with two separate neighborhoods, using complimentary targeted qPCR and metagenomics of wastewater before and after selective culture enrichment for clinically important Gram negatives. In total 26 ARG-type (1225 ARG-subtypes) were found across all samples, in which {beta}-lactam ARG was the richest (the number of different ARG-subtypes found) followed by multidrug, fluoroquinolone, macrolide-lincosamide-streptogramin (MLS) and aminoglycoside. The composition of ARGs in wastewater differed between raw wastewater pellets and culture-enriched wastewater samples and the resistomes clustered based on the type of location (Hospitals vs neighborhoods). Hospital wastewater was found to have higher diversity and greater abundance of ARGs compared to neighborhood wastewater when the composition profiles of ARGs in both raw and culture-enriched wastewater pellets. Clinically relevant ARG (i.e., VIM, NDM metallo-{beta}-lactamases) were detected in culture enrichment samples that were not identified in raw samples, despite a lower targeted sequencing depth. Wastewater-based surveillance is an effective, and potentially extremely important and powerful tool that could be developed to augment hospital-based infection control and antimicrobial stewardship programs, creating a safer space for those receiving care.

Areas of dense population congregation are prone to experience respiratory virus outbreaks. We monitored wastewater and clinic patients for the presence of respiratory viruses on a large, public university campus. Campus sewer systems were monitored in 16 locations for the presence of viruses using next generation sequencing over 22 weeks in 2023. During this period, we detected a surge in human adenovirus (HAdV) levels in wastewater. Hence, we initiated clinical surveillance at an on-campus clinic from patients presenting with acute respiratory infection. From whole genome sequencing of 123 throat and/or nasal swabs collected, we identified an outbreak of HAdV, specifically of HAdV-E4 and HAdV-B7 genotypes overlapping in time. The temporal dynamics and proportions of HAdV genotypes found in wastewater were corroborated in clinical infections. We tracked specific single nucleotide polymorphisms (SNPs) found in clinical virus sequences and showed that they arose in wastewater signals concordant with the time of clinical presentation, linking community transmission of HAdV to the outbreak. This study demonstrates how wastewater-based epidemiology can be integrated with surveillance at ambulatory healthcare settings to monitor areas prone to respiratory virus outbreaks and provide public health guidance. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=98 SRC="FIGDIR/small/24304990v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@f61580org.highwire.dtl.DTLVardef@1fdb62eorg.highwire.dtl.DTLVardef@1c3966aorg.highwire.dtl.DTLVardef@1c82efb_HPS_FORMAT_FIGEXP M_FIG C_FIG

The selection of appropriate viral indicators for evaluating wastewater treatment performance remains challenging because candidate markers have rarely been compared systematically within a unified analytical framework. Here, we collected influent and effluent samples monthly for one year from two wastewater treatment plants in Japan and conducted, to our knowledge, the first comprehensive comparison of 19 viral targets and one protozoan target using high-throughput quantitative PCR. Pepper mild mottle virus (PMMoV) was consistently detected at high concentrations, showed limited seasonal variability, and exhibited an approximately 1.0 log10 reduction, comparable to those observed for pathogenic viruses. In contrast, Carjivirus, formerly known as crAssphage, was present at the highest concentrations but showed significantly greater reduction than pathogenic viruses. Tomato brown rugose fruit virus (ToBRFV), despite its high abundance and emerging recognition as a potential marker, exhibited pronounced seasonal fluctuations. Other Tobamovirus species, such as cucumber green mottle mosaic virus and tobacco mild green mosaic virus, exhibited similar removal but lower prevalence compared with PMMoV. Overall, PMMoV demonstrated the most balanced performance in terms of abundance, stability, and removal behavior, supporting its use as a robust indicator for monitoring virus removal in wastewater treatment.

Nitrogen (N) pollution is a major threat to ecosystems and a driver of climate change through emissions of the greenhouse gas nitrous oxide (N2O). Mining activities are increasingly recognized for contributing to N pollution due to undetonated, N-based explosives. A woodchip denitrifying bioreactor, installed to treat nitrate-rich leachate from waste rock dumps in northern Sweden, was monitored for two years to determine the spatial and temporal distribution of microbial communities in pore water and woodchips and their genetic potential for different N transformation processes, and how this affected the N removal capacity and possible production of undesired N species, like ammonium, nitrite and N2O. About 80 and 65 % of the nitrate was removed from the leachate the first and second operational year, respectively, which agreed with a decrease in dissolved organic carbon in the outlet water. There was a succession in the microbial community over time and in space along the reactor length in both pore water and woodchips, which was reflected in the genetic potential for N cycling and ultimately also reactor performance. We conclude that DNRA had minimal impact on the overall N removal efficiency due to the low relative abundance of the key gene nrfA involved in DNRA and the low production of ammonium. However, nitrite, ammonium, and N2O were formed in the bioreactor and released in the effluent water, although direct emissions of N2O from the surface was low. The N2O production in the reactor might be explained by the ratio between the genetic potential for overall denitrification and N2O reduction in the woodchip and pore water communities, as indicated by the low ratio between the abundance of nir and nosZ genes. Altogether, the results indicate that the denitrification pathway was temporally as well as spatially separated along the reactor length, and that unwanted reactive N species were produced at different time points and locations in the reactor. Thus, the succession of microbial communities in woodchip denitrifying bioreactors treating mining impacted water develops slowly at low temperature, which impacts the reactor performance.

This study focused on the development of machine-learning- (ML) based strategies for mitigating nitrous oxide (N2O) emissions from various wastewater treatment systems in the United States measured using a benchmark USEPA-endorsed protocol. Results revealed that in general, poor process performance correlated with higher N2O emissions. Specifically, local variables including zone-specific dissolved oxygen, ammonia, and nitrite concentrations and global variables including effluent nitrite and nitrate concentrations contributed positively towards N2O emissions from both aerobic and anoxic zones of the process bioreactors. The optimal operational conditions identified for minimizing N2O emissions included operation of aerobic and anoxic zones at DO < 4 mg O2 L-1 and < 1 mg O2 L-1, respectively, coupled with appropriate solids retention times (SRTs) that maximize process performance. Accordingly, our results strongly underscore the utility of ML models in combination with bioprocess fundamentals for predicting and mitigating N2O emissions, while concomitantly optimizing wastewater treatment operations.

The uptake of extracellular electrons from cathodes by microbes is a recently discovered phenomenon. However, current knowledge on the diversity of microbes accepting extracellular electrons and populating biocathodes is scarce. Research is required to explore the distribution of extracellular electron uptake metabolism in the tree of life across microbial guilds. Here we characterize the electron uptake ability of microbial guilds enriched on H2, S2O32-, or CH4 and NH4+ from the same inoculum taken from a groundwater treatment sand-filter. We hypothesized that functional microbes having dense outer membrane cytochromes in their native pathway, such as pathways of NH4+, CH4, S2O32- and H2, can perform extracellular electron uptake. We aimed of addressing the following questions: (1) Are there any known microbial member of anticipated function performing extracellular electron transfer? (2) How does electron uptake efficiency vary between the different functional guilds? (3) How is the dissipated electron energy distributed across metabolisms? We developed and applied a novel pipeline to identify taxa utilizing direct electron energy and utilizing secondary microbial products. We report the putative direct electron uptake metabolism of types belonging to Methylomonas, UBA6140, and Nitrosomonas. Furthermore, members of Streptococcaceae, Rhizobiaceae, Streptococcus, Brevundimonas, Chryseobacterium, and Pseudomonas are detected as electroactive taxa. Our results reveal novel insights into the diversity, electrochemical activity, and metabolism of taxa performing direct electron uptake.

Tetrasphaera were recently identified based on the 16S rRNA gene as among the most abundant polyphosphate-accumulating organisms (PAOs) in global full-scale wastewater treatment plants (WWTPs) with enhanced biological phosphorus removal (EBPR). However, it is unclear how Tetrasphaera PAOs are selectively enriched in the context of the EBPR microbiome. In this study, an EBPR microbiome enriched with Tetrasphaera (accounting for 40% of 16S sequences on day 113) was built using a top-down design approach featuring multicarbon sources and a low dosage of allylthiourea. The microbiome showed enhanced nutrient removal (P removal ~85% and N removal ~80%) and increased P recovery (up to 23.2 times) compared with the seeding activated sludge from a local full-scale WWTP. The supply of 1 mg/L allylthiourea promoted the coselection of Tetrasphaera PAOs and Microlunatus PAOs and sharply reduced the relative abundance of both ammonia oxidizer Nitrosomonas and putative competitors Brevundimonas and Paracoccus, facilitating the establishment of the EBPR microbiome. Based on 16S rRNA gene analysis, a putative novel PAO species, EBPR-ASV0001, was identified with Tetrasphaera japonica as its closest relative. This study provides new knowledge on the establishment of a Tetrasphaera-enriched microbiome facilitated by allylthiourea, which can be further exploited to guide future process upgrading and optimization to achieve and/or enhance simultaneous biological phosphorus and nitrogen removal from high-concentration wastewater.

Nutrient-rich food-processing wastewaters represent valuable yet under-utilised side streams for sustainable protein production in the form of microbial biomass. Here we present an integrated dual-loop bioprocess that converts soybean-processing wastewater into microbial single-cell protein (SCP) while achieving substantial nutrient removal and product refinement. In the first loop, previously enriched microbial consortia were inoculated and cultivated in four parallel sequencing batch reactors (SBRs) for 44days at a hydraulic retention time (HRT) of 3days. This bioprocess configuration demonstrated features that support future scale-up while maintaining process stability, achieving a protein content of 33.3{+/-}3.2%, doubling the protein yield (15.32{+/-}3.49g dry weight per g soluble TKN) and quadrupling the production rate (0.29{+/-}0.06g dry weight L-1 d-1) compared to operating reactors without inoculation (HRT: 7.2days). Effluent treatment was stable, with 84% carbon and 78% nitrogen removal efficiencies, demonstrating efficient nutrient recovery. The SCP biomass was enriched in functional taxa, including Acidipropionibacterium, Lactococcus, Megasphaera, and Azospirillum, suggesting that reactor conditions and inoculum selection promoted a stable, protein-productive microbial community with potential probiotic benefits. In the second loop, bioreactor effluent was reused as aqueous matrix for heat treatment (60{degrees}C) of the SCP biomass, reducing the RNA content from 8.6% to 2.6%, with a 39% biomass loss accompanied by a 30% increase in total amino acid concentration. Hence, our valorisation approach integrates microbial biomass production, effluent reuse, and product refinement within a circular framework. The system provides a resource-efficient pathway for converting food-sector side streams into high-quality microbial community-based SCP, highlighting its potential scalability for sustainable nutrient and water management.

Pharmaceutical compounds (PhC) are an important environmental issue, because of their high variety, potentially toxic byproducts and bioactivity at low concentrations. PhC concentrations in WWTP effluents often exhibit large and rapid variations that are difficult to record. Passive samplers are helpful to incorporate spot pollution events and register PhC occurrence at low concentrations. In this work, we aim at (i) studying PhC accumulation in polar organic chemical integrative samplers (POCIS) and environmental biofilms exposed to urban (U) and hospital (H) treated effluents and (ii) evaluating the capacity of POCIS to predict changes in biofilm microbiome over a defined time period. Moreover, we (iii) determine the enrichment of PhC in the recipient river to evaluate levels of environmental contamination and potential effects on microbial biofilms. Biofilms and POCIS were installed in treated effluents and in the recipient river to measure the accumulation of PhC. In parallel, microbial biofilm communities were studied using DNA metabarcoding. The duration of each deployment was one month and the experiment was repeated six times. The performance of POCIS and biofilms to quantify PhC was depending on the compound. POCIS appeared well adapted to reveal contamination trends similar to these in the water column and to identify key PhC drivers of microbial changes. POCIS have the potential to predict pharmaceutical effects on biofilm community structure.

Central Michigan University (CMU) participated in a state-wide SARS-CoV-2 wastewater monitoring program since 2021. Wastewater samples were collected from on-campus sites and nine off-campus wastewater treatment plants servicing small metropolitan and rural communities. SARS-CoV-2 genome copies were quantified using droplet digital PCR and results were reported to the health department. One rural, off-campus site consistently produced higher concentrations of SARS-CoV-2 genome copies. Samples from this site were sequenced and initially contained predominately Alpha variant lineage Q.3, which transitioned to lineage Q.4. Alpha variant lineage Q.3/Q.4 was detected at this site beginning in fall 2021 and continued until summer 2023. Mutational analysis of reconstructed genes revealed divergence from the Alpha variant lineage Q.3 clinical sequence over time, including numerous mutations in the surface glycoprotein RBD and NTD. We discuss the possibility that a chronic SARS-CoV-2 infection accumulated adaptive mutations that promoted long-term infection. This study reveals that small wastewater treatment plants can enhance resolution of rare events and facilitate reconstruction of viral genomes due to the relative lack of contaminating sequences.