Influent Wastewater Research Articles

Reduction of SARS-CoV-2 by biological nutrient removal and disinfection processes in full-scale wastewater treatment plants

Detection of SARS-CoV-2 RNA in wastewater poses people's concerns regarding the potential risk in water bodies receiving wastewater treatment effluent, despite the infectious risk of SARS-CoV-2 in wastewater being speculated to be low. Unlike well-studied nonenveloped viruses, SARS-CoV-2 in wastewater is present abundantly in both solid and liquid fractions of wastewater. Reduction of SARS-CoV-2 in past studies were likely underestimated, as SARS-CoV-2 in influent wastewater were quantified in either solid or liquid fraction only. The objectives of this study were (i) to clarify the reduction in SARS-CoV-2 RNA during biological nutrient removal and disinfection processes in full-scale WWTPs, considering the SARS-CoV-2 present in both solid and liquid fractions of wastewater, and (ii) to evaluate applicability of pepper mild mottle virus (PMMoV) as a performance indicator for reduction of SARS-CoV-2 in WWTPs. Accordingly, large amount of SARS-CoV-2 RNA were partitioned in the solid fraction of influent wastewater for composite sampling than grab sampling. When SARS-CoV-2 RNA in the both solid and liquid fractions were considered, log reduction values (LRVs) of SARS-CoV-2 during step-feed multistage biological nitrogen removal (SM-BNR) and enhanced biological phosphorus removal (EBPR) processes ranged between>2.1–4.4 log and did not differ significantly from those in conventional activated sludge (CAS). The LRVs of SARS-CoV-2 RNA in disinfection processes by ozonation and chlorination did not differ significantly. PMMoV is a promising performance indicator to secure reduction of SARS-CoV-2 in WWTPs, because of its higher persistence in wastewater treatment processes and abundance at a detectable concentration even in the final effluent after disinfection.

Enhancement of a Decentralized Wastewater Treatment System by Applying a Bio-tower - A Project for Improving our Environmental Footprint

To investigate if decentralized wastewater treatment systems impact on the environment performance can be improved, a Field Bio-tower Tank Septic system was designed, installed on an existing 1,500-gal (5678 l) decentralized wastewater treatment system. The system was started up during a 14-day start-up phase, followed by a 130-day test phase. The system was operated at an average hydraulic retention time of 16.7 days based on a 90 gal (204.4 l) daily influent wastewater flow.
 The systems recirculation flow for the bio-tower was 22.6 l/min (6 gal/min) for the start-up phase and 40.0 l/min (10.6 gal/min) for the test period with a corresponding recirculation of the entire septic tank from 5.7 to 10.1 times a day respectively.
 During the testing period a daily average temperature of 19.3°C (66.7°F) and an average rainfall of 5 l/m² was observed. The FBST system operated at an average influent pH level and temperature of 8.6 and 22.0°C. The average effluent pH and temperature was 8.5 of 20.9°C.
 The installed system reduced on average the influent TS from 79151 mg/l to 43021 mg/l and showed an average reduction of the TDS from 36424 mg/l to 21965 mf/l. The TSS and VSS was reduced on average from 94.1 mg/l to 69.9 mg/l and 89.0 mg/l to 64.9 mg/l.
 NH3-N content was reduced from 57.5 mg/l in the influent to 53.4 mg/l in the effluent. The average BOD and COD was reduced from the average influent value of 88.2 mg/l and 318.2 mg/l to 66.8 mg/l and 256.2 mg/l respectively. However, at the end of the 130-day test period BOD and COD values were the lowest at 15.5 mg/l and 56.0 mg/l respectively, indicating bacteria cultures are well established and the system operation of the FBTS system is mostly constant.
 This research showed that the FBST system can improve operations of a DWWTS system significantly reducing the effluent load over 10 times compared to the influent load of the system.

The influence of military actions on operation conditions of municipal wastewater treatment plants (MWWTPs) of Kharkiv city

The events related to the military aggression against Ukraine had a significant impact on the operation conditions of the municipal wastewater treatment plants (MWWTPs) of Kharkiv city. The quantitative and qualitative indicators of influent wastewater of municipal wastewater treatment plants #1 and #2 - the biological treatment complexes "Dykanivskyi" (MWWTP-1) and "Bezlyudivskyi" (MWWTP-2) significantly had changed because of reduction of the city's population and the reduction of its industrial potential. The daily wastewater pollution loads on wastewater treatment plants in the period from March 2022 to March 2023 had decreased by almost half compared to pre-war times. The actual daily wastewater discharges were: at MWWTP-1 – 216.8 thousand m³/day (or 54.2% of design capacity according to Project of reconstruction of wastewater treatment plants and the sludge treatment line); at MWWTP-2 – 69.0 thousand m³/day (or 43.2%, respectively). The actual daily load of wastewater pollution during military actions was on average (as a percentage of the design value): at the MWWTP-1 by COD - 60%; by BOD5 – 52%; by TSS – 51%; by Ammonium Nitrogen - 66%; and by Phosphates – 27%; at MWWTP-2 by COD - 52%; by BOD5 – 51%; by TSS - 30%; by Ammonium Nitrogen - 64%; and by Phosphates – 43%. The negative consequences of termination of the started works on the reconstruction of wastewater treatment plants due to military actions on their operation conditions are considered in the paper. The conclusion was made about the need to make corrections in the project of reconstruction of wastewater treatment plants and sludge treatment in case of its restoration, taking into account the actual situation.

Evaluating resource recovery potential and process feasibility of direct membrane ultrafiltration of municipal wastewater at demonstration scale

The aim of this work was to assess the feasibility of applying ultrafiltration membranes for the direct filtration of municipal wastewater. The effect of different influents (raw wastewater and the primary settler effluent of a municipal wastewater treatment plant) and operating solids concentration (about 1, 2.6, 6 and 11 g L−1) was evaluated. Increasing the operating solids concentration sharply reduced membrane fouling, achieving a minimum of about 0.55 mbar per day when operating above 6 g L−1. Filtering raw municipal wastewater also had a beneficial effect on the filtration performance, although only when operating at a low solids concentration (under 2.6 g L−1). High quality permeates were obtained regardless of operating conditions, meeting European discharge requirements for no sensible environments. High resource recoveries were achieved thanks to the direct filtration of wastewater, capturing about 80-85 and 20%–40% of the influent COD and nutrients, respectively. The energy and carbon footprint balances showed promising results, achieving energy recoveries and carbon footprints of about 0.46-0.40 kWh per m3 of influent municipal wastewater and -[0.19-0.16] kg CO2-eq per m3 of influent municipal wastewater when considering the process energy demands and potential energy recovery. Additionally, profits can be obtained from MWW treatment by this alternative, achieving about €0.035–0.021 per m3 of influent MWW when operating at a flux of 10 LMH which entail a payback period for the initial membrane acquisition of 12.3–20.1 years.

Transition of antimicrobial resistome in wastewater treatment plants: impact of process configuration, geographical location and season

Antimicrobial resistome in wastewater treatment plants was investigated via shotgun metagenomic analysis over a variety of geographical locations, seasons, and biological treatment configurations. The results revealed that the transition of the antimicrobial resistome occurred at two locations during wastewater treatment, which resulted in a distinctive antimicrobial resistome in influent wastewater, activated sludge, and treated effluent. The antimicrobial resistome in influent wastewater was characterized by a high abundance of antibiotic resistance genes (ARGs) on clinically important drugs, whereas sludge retained a higher abundance of multidrug ARGs associated with efflux pump. Seasonality was the primary factor affecting antimicrobial resistome in influent wastewater, which partially succeeded to the subsequent resistome of activated sludge and treated effluent. Importantly, some ARGs on clinically important drugs in influent wastewater passed through the biological treatment to be discharged in the treated effluent, except in the membrane bioreactor process.

The Reduction of SARS-CoV-2 RNA Concentration in the Presence of Sewer Biofilms

Wastewater surveillance has been widely used to track the prevalence of SARS-CoV-2 in communities. Although some studies have investigated the decay of SARS-CoV-2 RNA in wastewater, understanding about its fate during wastewater transport in real sewers is still limited. This study aims to assess the impact of sewer biofilms on the dynamics of SARS-CoV-2 RNA concentration in naturally contaminated real wastewater (raw influent wastewater without extra SARS-CoV-2 virus/gene seeding) using a simulated laboratory-scale sewer system. The results indicated that, with the sewer biofilms, a 90% concentration reduction of the SARS-CoV-2 RNA was observed within 2 h both in wastewater of gravity (GS, gravity-driven sewers) and rising main (RM, pressurized sewers) sewer reactors. In contrast, the 90% reduction time was 8–26 h in control reactors without biofilms. The concentration reduction of SARS-CoV-2 RNA in wastewater was significantly more in the presence of sewer biofilms. In addition, an accumulation of c.a. 260 and 110 genome copies/cm2 of the SARS-CoV-2 E gene was observed in the sewer biofilm samples from RM and GS reactors within 12 h, respectively. These results confirmed that the in-sewer concentration reduction of SARS-CoV-2 RNA in wastewater was likely caused by the partition to sewer biofilms. The need to investigate the in-sewer dynamic of SARS-CoV-2 RNA, such as the variation of RNA concentration in influent wastewater caused by biofilm attachment and detachment, was highlighted by the significantly enhanced reduction rate of SARS-CoV-2 RNA in wastewater of sewer biofilm reactors and the accumulation of SARS-CoV-2 RNA in sewer biofilms. Further research should be conducted to investigate the in-sewer transportation of SARS-CoV-2 and their RNA and evaluate the role of sewer biofilms in leading to underestimates of COVID-19 prevalence in communities.

High-risk antibiotics positively correlated with antibiotic resistance genes in five typical urban wastewater

Antibiotic resistance genes (ARGs) and antibiotic amount increased within close proximity to human dominated ecosystems. However, few studies assessed the distribution of antibiotics and ARGs in multiple ecosystems especially the different urban wastewater. In this study, the spatial distribution of ARGs and antibiotics across the urban wastewater included domestic, livestock, hospital, pharmaceutical wastewater, influent of the wastewater treatment plant (WWTP) in Northeast China. The q-PCR results showed that ARGs were most abundant in community wastewater and followed by WWTP influent, livestock wastewater, pharmaceutical wastewater and hospital wastewater. The ARG composition differed among the five ecotypes with qnrS was the dominant ARG subtypes in WWTP influent and community wastewater, while sul2 dominant in livestock, hospital, pharmaceutical wastewater. The concentration of antibiotics was closely related to the antibiotic usage and consumption data. In addition to the high concentration of azithromycin at all sampling points, more than half of the antibiotics in livestock wastewater were veterinary antibiotics. However, antibiotics that closely related to humankind such as roxithromycin and sulfamethoxazole accounted for a higher proportion in hospital wastewater (13.6%) and domestic sewage (33.6%), respectively. The ambiguous correlation between ARGs and their corresponding antibiotics was detected. However, antibiotics that exhibited high ecotoxic effects were closely and positively correlated with ARGs and the class 1 integrons (intI1), which indicated that high ecotoxic compounds might affect antimicrobial resistance of bacteria by mediating horizontal gene transfer of ARGs. The coupling mechanism between the ecological risk of antibiotics and bacterial resistance needed to be further studied, and thereby provided a new insight to study the impact of environmental pollutants on ARGs in various ecotypes.

Establishment of wastewater-based SARS-CoV-2 monitoring system over two years: Case studies in South Korea

With the global COVID-19 pandemic, wastewater surveillance has received a considerable attention as a method for the early identification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in wastewater treatment plant (WWTP) and sewer systems. For the first time in Korea, this study utilized the wastewater surveillance technique to monitor the COVID-19 outbreak. Sampling efforts were carried out at the WWTPs in the capital city of Korea, Seoul, and Daegu the place where the first severe outbreak was reported. The RNA of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been extracted from the collected wastewater influent and primary sewage sludge samples. The outcomes were contrasted with the COVID-19 cases in the WWTPs served area. Additionally, whole transcriptome sequencing was used to compare the microbial community alterations before and after the COVID-19 outbreak and SARS-CoV-2 variations. The results demonstrated that the changes in SARS-CoV-2 RNA concentrations in the influent and sludge matched the trends of reported COVID-19 cases, especially sludge showed high-resolution data, which is well-matched when fewer COVID-19 cases (0−250) are reported. Interestingly, one month before the clinical report, we found that the SARS-CoV-2 Beta variant (South Africa, B.1.351) in the wastewater. In addition, the Aeromonas bacterial species was dominated (21.2%) among other bacterial species in wastewater after the COVID-19 outbreak, suggesting a potential indirect microbial indicator of the COVID-19 outbreak.

The knock-on effects of different wastewater feeding modes: Change in microbial communities versus resistance genes in pilot-scale aerobic sludge granulation reactors

To explore the effects of wastewater feeding modes on the formation of aerobic granular sludge (AGS) and the complex relationships between resistance genes and bacteria, two pilot-scale sequencing batch reactors (SBRs) were established. The SBR with influent wastewater introduced uniformly through pipes at bottom was designated as BSBR, and the SBR with inlet wastewater flowing directly from top was TSBR. BSBR formed dense AGS due to uniform wastewater feeding at bottom, while TSBR failed to cultivate AGS. Metagenomic sequencing illustrated that rapid growth of AGS in BSBR was accompanied with increase of antibiotic resistance genes (ARGs) abundance, but ARGs diminished when the size of AGS was stable. The ARGs continued to elevate in TSBR, and abundance of metal resistance genes (MRGs) was always higher than that in BSBR. Two reactors had markedly different bacterial community, microbes in BSBR owned stronger activity, conferred greater potential to proliferate. AdeF in two systems had the most complex gene-bacteria relationships which would undergo HGT within bacterial genus. The different feeding modes of wastewater directly led to the changing size of sludge, which caused knock-on effects of variations in the abundance of microbial communities and resistance genes. This study provided promising suggestions for the rapid cultivation of AGS and control of resistance genes at pilot-scale.

Copper-modified carbon nano-onions as electrode modifiers for the electroanalysis of the antiretroviral drug Efavirenz

The high prescription and consumption rate of antiretroviral drugs (ARV) such as Efavirenz (EFV) in South Africa for the treatment of the human immunodeficiency virus (HIV) has resulted in its presence in wastewater and surface water. Herein we report the electroanalysis of EFV at oxidised boron-nitrogen doped carbon nano-onions (oxi-BNCNO) and microscale branched copper cluster (CuC) modified glassy carbon electrodes. Potentiostatic electrodeposition of CuC on the oxi-BNCNO/GCE platform resulted in a stable and electrocatalytic surface that accelerated electron transfer between the analyte and the CuC/oxi-BNCNO/GCE surface, making quantification efficient. The electroactive surface area of CuC/oxi-BNCNO/GCE was estimated as being 3 times higher than bare GCE and twice that of oxi-BNCNO/GCE. The electrooxidation of EFV on a CuC/oxi-BNCNO/GCE sensor resulted in a pH-dependant anodic peak in the potential range of 0.8 to 1.2 V vs Ag/AgCl (3M KCl). The EFV voltammetric signal increased linearly with increasing concentration of EFV in the linear dynamic range (LDR) of 0.01 – 1.0 µM and 0.5 – 20 µM with a limit of detection (LOD) and quantification (LOQ) of 1.2 and 3.97 nM, respectively. Moreover, the sensor had a sensitivity of 23 µA ∙ cm−2 ∙ µM−1 and was selective to 100-fold of interferents including heavy metal ions and other ARVs with the exception of high concentrations of nevirapine. The developed electroanalytical method was successfully applied for the determination of EFV in real samples such as wastewater influent and effluent, drinking/tap water, and a pharmaceutical formulation with recovery ranging from 97.8% to 109.5%.

Nonbacterial Microflora in Wastewater Treatment Plants: an Underappreciated Potential Source of Pathogens.

Wastewater treatment plants (WWTPs) receive and treat large volumes of domestic, industrial, and urban wastewater containing pathogenic and nonpathogenic microorganisms, chemical compounds, heavy metals, and other potentially hazardous substances. WWTPs play an essential role in preserving human, animal, and environmental health by removing many of these toxic and infectious agents, particularly biological hazards. Wastewater contains complex consortiums of bacterial, viral, archaeal, and eukaryotic species, and while bacteria in WWTP have been extensively studied, the temporal and spatial distribution of nonbacterial microflora (viruses, archaea, and eukaryotes) is less understood. In this study, we analyzed the viral, archaeal, and eukaryotic microflora in wastewater throughout a treatment plant (raw influent, effluent, oxidation pond water, and oxidation pond sediment) in Aotearoa (New Zealand) using Illumina shotgun metagenomic sequencing. Our results suggest a similar trend across many taxa, with an increase in relative abundance in oxidation pond samples compared to influent and effluent samples, except for archaea, which had the opposite trend. Additionally, some microbial families, such as Podoviridae bacteriophages and Apicomplexa alveolates, appeared largely unaffected by the treatment process, with their relative abundance remaining stable throughout. Several groups encompassing pathogenic species, such as Leishmania, Plasmodium, Toxoplasma, Apicomplexa, Cryptococcus, Botrytis, and Ustilago, were identified. If present, these potentially pathogenic species could be a threat to human and animal health and agricultural productivity; therefore, further investigation is warranted. These nonbacterial pathogens should be considered when assessing the potential for vector transmission, distribution of biosolids to land, and discharge of treated wastewater to waterways or land. IMPORTANCE Nonbacterial microflora in wastewater remain understudied compared to their bacterial counterparts despite their importance in the wastewater treatment process. In this study, we report the temporal and spatial distributions of DNA viruses, archaea, protozoa, and fungi in raw wastewater influent, effluent, oxidation pond water, and oxidation pond sediments by using shotgun metagenomic sequencing. Our study indicated the presence of groups of nonbacterial taxa which encompass pathogenic species that may have potential to cause disease in humans, animals, and agricultural crops. We also observed higher alpha diversity in viruses, archaea, and fungi in effluent samples than in influent samples. This suggests that the resident microflora in the wastewater treatment plant may be making a greater contribution to the diversity of taxa observed in wastewater effluent than previously thought. This study provides important insights to better understand the potential human, animal, and environmental health impacts of discharged treated wastewater.

Sorption affinity and mechanisms of per-and polyfluoroalkyl substances (PFASs) with commercial sorbents: Implications for passive sampling

Effective monitoring tools, including passive samplers, are essential for the wide range of per- and polyfluoroalkyl substances (PFASs) in aquatic matrices. However, knowledge of the extent and mechanisms of PFASs sorption with sorbents in a passive sampling context is limited. To address this, sorption behavior of 45 anionic, neutral and zwitterionic PFASs ranging in perfluorocarbon chain length (C3-C16) and functional groups with 11 different commercial sorbents (cross-linked β-cyclodextrin polymers, activated carbon, anion exchange (AE), cation exchange, hydrophilic-lipophilic balanced (HLB) and non-polar) was investigated. A broad range of equilibrium sorbent-MilliQ water (MQ) distribution coefficients (Kd) were observed (10−1.95 to 108.30 mL g−1). Similar sorbent types (e.g., various AE and HLB sorbents) exhibited very different sorption behavior, likely due to their different polymeric structures and relative importance of sorbate/sorbent interactions other than coulombic interactions. HLB and AE with hydroxyl functionalities are most effective for sampling of the full suite of PFASs. Reduced sorptive affinity was observed in the presence of matrix co-constituents in wastewater influent for most PFASs. HLB had the smallest reduction in log Kd in wastewater suggesting that these sorbents are appropriate for applications in complex matrices. Sufficient sorbent capacity was observed for linear uptake of many target analytes which facilitates passive sampling.

Nitrite pathway in A2/O WWTPs: Modelling organic matter reduction, operational cost and N2O emissions

Deciding whether the implementation of the nitrite pathway is useful for the treatment of a given wastewater depends on several criteria such as organic matter availability, N and P removal, N2O emissions and operational costs. This work is a simulation-based study with a conventional Anaerobic/Anoxic/Oxic (A2/O) WWTP where the nitrite pathway can be implemented. The outcomes are general correlations to calculate the minimum COD requirements for a certain influent and a practical decision tree on the opportunities of nitrite pathway in A2/O WWTPs as a function of the influent wastewater composition (for P and N concentrations in the ranges 3–11 mgPO4−3-P·L−1 and 20–60 mg NH4+-N·L−1). This study shows that the implementation of the nitrite pathway reduces the COD requirements (depending on the influent, between 9 and 68%). It also can lead to a reduction in aeration costs. For an equivalent COD in the influent, the implementation of the nitrite pathway compared to the nitrate pathway results in a reduction of 41-47% in aeration costs and similar sludge production, leading to a reduction in the operational cost index of 10-16%. However, it is essential to note that this strategy can also lead to increased N2O emissions, with an emission factor for the nitrite pathway in the range of 2.5–17 times that of the nitrate pathway.

Identification, occurrence, concentration and composition profile of fiproles in municipal wastewater treatment plants

Although fipronil and several of its transformation products are ubiquitous in aquatic environments, limited information is available on the structural identities, detection frequencies, concentrations and composition profiles of fiproles (fipronil and its known and unknown transformation products) in municipal wastewater treatment plants (WWTPs). In this study, a suspect screening analysis was applied to identify and characterize fipronil transformation products in 16 municipal WWTPs from three cities in China. In addition to fipronil and its four transformation products (fipronil amide, fipronil sulfide, fipronil sulfone and desulfinyl fipronil), fipronil chloramine and fipronil sulfone chloramine were detected for the first time in municipal wastewater. Moreover, the cumulative concentrations of six transformation products were 0.236 ng/L and 3.44 ng/L in wastewater influents and effluents, and accounted for one-third (in influents) to half (in effluents) of fiproles. Of those transformation products, two chlorinated byproducts (fipronil chloramine and fipronil sulfone chloramine) were major transformation products in both municipal wastewater influents and effluents. Notably, the log Kow and bioconcentration factor (evaluated by EPI Suite software) of fipronil chloramine (log Kow = 6.64, and BCF = 11,200 L/kg wet-wt) and fipronil sulfone chloramine (log Kow = 4.42, and BCF = 382.9 L/kg wet-wt) were greater than that of their parent compound. Considering the persistence, bioaccumulation potential and toxicity, the high detection rates of fipronil chloramine and fipronil sulfone chloramine in urban aquatic systems need to be specifically considered in future ecological risk assessments.

Phenotypic and genotypic characteristics of beta-lactamase dominant with CARBA, AmpC, and ESBL-producing bacteria in municipal wastewater influent in Helsinki, Finland

Analysing samples of municipal wastewater influent (before treatment) can help to map the status of antibiotic-resistant bacteria (ARB) at the population level in sewershed communities and may also help in predicting the public health risks of ARB in surface water because of the outfall of wastewater. In this study, we investigated the bacterial isolates carrying beta-lactamase genes in wastewater and compared their genotypic and phenotypic characteristics. A total of 399 bacterial isolates grown on CHROMagarESBL (n=207) and CHROMagarKPC (n=192) from composite wastewater influent samples (n=7) from the Viikinmäki wastewater treatment plant (Helsinki) were subcultured, nucleic acid was extracted, and the prevalence of different beta-lactamase genes was screened with multiplex polymerase chain reaction (PCR). All PCR-positive isolates were identified with MALDI-TOF. A total of 32.6% of isolates (130 of 399) were PCR positive for at least one resistance gene, and 13% of these positive isolates out of 130 had at least three resistance genes. Among the 22 detected genes, blaGES group was the most prevalent, at 25.8% (n=198; many isolates carried multiple genes), followed by blaMOX (13.1%) and blaTEM (10.1%) as most frequently detected. Furthermore, out of 18 different bacterial species/genera detected as carrying beta-lactamase genes, A. hydrophila/caviae (28.5%), Enterobacter spp. (16.9%), and E. coli (14.6%) were the most prevalent. Enterobacter spp., Aeromonas spp., and K. cryocescens potentially carried AmpC genes, and E. coli carried ESBL genes. We recorded a huge variety of beta-lactamases (blaAmpC, blaESBL, and blaCARBA) genes in many potential pathogens that probably originated from both enteric and environmental sources.

First quantitative detection of tomato brown rugose fruit virus in wastewater in Louisiana

We investigated the occurrence of tomato brown rugose fruit virus (ToBRFV) at a conventional wastewater treatment plant in Louisiana over a 13-month period, from March 2017 to March 2018. Influent, secondary effluent, and final effluent wastewater samples were collected monthly, and viruses were concentrated by the adsorption-elution method using an electronegative filter, followed by the detection using quantitative polymerase chain reaction. ToBRFV was detected in 10 (77 %) of 13 influent samples, 9 (69 %) of 13 in secondary effluent, and 6 (50 %) of 12 final effluents. The concentrations of ToBRFV in the influent samples ranged from 3.5 to 6.1 log10 copies/L and it was always higher than those in secondary or final effluents. Wastewater samples showed a high positive ratio of ToBRFV during fall and winter months. The findings highlight that routine monitoring of new viral indicator such as ToBRFV is necessary to understand its environmental distribution and correlation with pathogenic viruses. This is the first study providing quantitative data on the occurrence of ToBRFV in wastewater.

High-resolution characterization of short-term temporal variability in the taxonomic and resistome composition of wastewater influent.

Wastewater-based epidemiology (WBE) for population-level surveillance of antimicrobial resistance (AMR) is gaining significant traction, but the impact of wastewater sampling methods on results is unclear. In this study, we characterized taxonomic and resistome differences between single-timepoint-grab and 24 h composites of wastewater influent from a large UK-based wastewater treatment work [WWTW (population equivalent: 223 435)]. We autosampled hourly influent grab samples (n=72) over three consecutive weekdays, and prepared additional 24 h composites (n=3) from respective grabs. For taxonomic profiling, metagenomic DNA was extracted from all samples and 16S rRNA gene sequencing was performed. One composite and six grabs from day 1 underwent metagenomic sequencing for metagenomic dissimilarity estimation and resistome profiling. Taxonomic abundances of phyla varied significantly across hourly grab samples but followed a repeating diurnal pattern for all 3 days. Hierarchical clustering grouped grab samples into four time periods dissimilar in both 16S rRNA gene-based profiles and metagenomic distances. 24H-composites resembled mean daily phyla abundances and showed low variability of taxonomic profiles. Of the 122 AMR gene families (AGFs) identified across all day 1 samples, single grab samples identified a median of six (IQR: 5-8) AGFs not seen in the composite. However, 36/36 of these hits were at lateral coverage <0.5 (median: 0.19; interquartile range: 0.16-0.22) and potential false positives. Conversely, the 24H-composite identified three AGFs not seen in any grab with higher lateral coverage (0.82; 0.55-0.84). Additionally, several clinically significant human AGFs (bla VIM, bla IMP, bla KPC) were intermittently or completely missed by grab sampling but captured by the 24 h composite. Wastewater influent undergoes significant taxonomic and resistome changes on short timescales potentially affecting interpretation of results based on sampling strategy. Grab samples are more convenient and potentially capture low-prevalence/transient targets but are less comprehensive and temporally variable. Therefore, we recommend 24H-composite sampling where feasible. Further validation and optimization of WBE methods is vital for its development into a robust AMR surveillance approach.

Antibiotic resistance gene sequencing is necessary to reveal the complex dynamics of immigration from sewers to activated sludge.

Microbial community composition has increasingly emerged as a key determinant of antibiotic resistance gene (ARG) content. However, in activated sludge wastewater treatment plants (AS-WWTPs), a comprehensive understanding of the microbial community assembly process and its impact on the persistence of antimicrobial resistance (AMR) remains elusive. An important part of this process is the immigration dynamics (or community coalescence) between the influent and activated sludge. While the influent wastewater contains a plethora of ARGs, the persistence of a given ARG depends initially on the immigration success of the carrying population, and the possible horizontal transfer to indigenously resident populations of the WWTP. The current study utilized controlled manipulative experiments that decoupled the influent wastewater composition from the influent microbial populations to reveal the fundamental mechanisms involved in ARG immigration between sewers and AS-WWTP. A novel multiplexed amplicon sequencing approach was used to track different ARG sequence variants across the immigration interface, and droplet digital PCR was used to quantify the impact of immigration on the abundance of the targeted ARGs. Immigration caused an increase in the abundance of over 70 % of the quantified ARGs. However, monitoring of ARG amplicon sequence variants (ARG-ASVs) at the immigration interface revealed various immigration patterns such as (i) suppression of the indigenous mixed liquor ARG-ASV by the immigrant, or conversely (ii) complete immigration failure of the influent ARG-ASV. These immigration profiles are reported for the first time here and highlight the crucial information that can be gained using our novel multiplex amplicon sequencing techniques. Future studies aiming to reduce AMR in WWTPs should consider the impact of influent immigration in process optimisation and design.

Optimised protocol for monitoring SARS-CoV-2 in wastewater using reverse complement PCR-based whole-genome sequencing.

Monitoring the spread of viral pathogens in the population during epidemics is crucial for mounting an effective public health response. Understanding the viral lineages that constitute the infections in a population can uncover the origins and transmission patterns of outbreaks and detect the emergence of novel variants that may impact the course of an epidemic. Population-level surveillance of viruses through genomic sequencing of wastewater captures unbiased lineage data, including cryptic asymptomatic and undiagnosed infections, and has been shown to detect infection outbreaks and novel variant emergence before detection in clinical samples. Here, we present an optimised protocol for quantification and sequencing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in influent wastewater, used for high-throughput genomic surveillance in England during the COVID-19 pandemic. This protocol utilises reverse compliment PCR for library preparation, enabling tiled amplification across the whole viral genome and sequencing adapter addition in a single step to enhance efficiency. Sequencing of synthetic SARS-CoV-2 RNA provided evidence validating the efficacy of this protocol, while data from high-throughput sequencing of wastewater samples demonstrated the sensitivity of this method. We also provided guidance on the quality control steps required during library preparation and data analysis. Overall, this represents an effective method for high-throughput sequencing of SARS-CoV-2 in wastewater which can be applied to other viruses and pathogens of humans and animals.

Three years of wastewater surveillance for new psychoactive substances from 16 countries

The proliferation of new psychoactive substances (NPS) over recent years has made their surveillance complex. The analysis of raw municipal influent wastewater can allow a broader insight into community consumption patterns of NPS. This study examines data from an international wastewater surveillance program that collected and analysed influent wastewater samples from up to 47 sites in 16 countries between 2019 and 2022. Influent wastewater samples were collected over the New Year period and analysed using validated liquid chromatography – mass spectrometry methods. Over the three years, a total of 18 NPS were found in at least one site. Synthetic cathinones were the most found class followed by phenethylamines and designer benzodiazepines. Furthermore, two ketamine analogues, one plant based NPS (mitragynine) and methiopropamine were also quantified across the three years. This work demonstrates that NPS are used across different continents and countries with the use of some more evident in particular regions. For example, mitragynine has highest mass loads in sites in the United States, while eutylone and 3-methylmethcathinone increased considerably in New Zealand and in several European countries, respectively. Moreover, 2F-deschloroketamine, an analogue of ketamine, has emerged more recently and could be quantified in several sites, including one in China, where it is considered as one of the drugs of most concern. Finally, some NPS were detected in specific regions during the initial sampling campaigns and spread to additional sites by the third campaign. Hence, wastewater surveillance can provide an insight into temporal and spatial trends of NPS use.