Scattered and transmitted light as surrogates for activated carbon residual in advanced wastewater treatment processes: Investigating the influence of particle size.

The use of powdered activated carbon (PAC) is a common process in advanced wastewater treatment to remove micropollutants. Retention and separation of PAC is essential as PAC loaded with micropollutants should not be released into the environment. Determining the activated carbon (AC) residual in the effluent poses a challenge, as there is currently no on-line measurement method. In this study, the correlation between turbidity, measured by scattered light, and absorption at wavelength of 550 nm (Absorption550 nm), measured by transmitted light, was investigated in relation to the AC residue. Linear correlations for turbidity (R2 = 0.95) and Absorption550 nm (R2 = 1.00) to AC concentrations were observed in both laboratory and full-scale experiments in a pilot plant where superfine PAC was added prior to Pile Cloth Media Filtration (PCMF). Decreasing the particle size (d50) while maintaining the same AC concentration leads to increased turbidity: Therefore, a fourfold reduction in d50 results in a 2- to 3-fold increase in turbidity, whereas a 30-fold reduction in d50 leads to a 6-to 8-fold increase. Furthermore, the original wastewater turbidity led to a parallel shift in the linear correlation between turbidity and AC. Coagulant doses of up to 400 mg Me3+/g AC resulted in a 50% reduction in turbidity. However, higher concentrations from 400 to 1,000 mg Me3+/g AC resulted in increased turbidity with only a 30% reduction compared to the initial turbidity. The study also highlights the significance of AC particle size in optical measurements, impacting result accuracy.

Looking deeper into the effects of scouring and aeration on membrane aerated biofilms: Analysis of nitrogen conversion, oxygen profiles and nitrous oxide emissions.

This study investigated the effects of aeration and scouring strategies on the performance of Membrane Aerated Biofilm Reactors (MABRs) and the distribution of oxygen and nitrous oxide in the biofilm. Four flat sheet MABRs were operated with synthetic feed under different conditions: two with intermittent aeration (iMABR) and two with continuous aeration (cMABR). Scouring was induced by bubbling dinitrogen gas through the reactor bulk at low and high frequencies (LF and HF). In the iMABRs, a partial nitritation biofilm initially developed, but the biofilm adapted to the aeration strategy over time and became nitrifying. The cMABRs directly developed a nitrifying biofilm without a significant phase of partial nitritation. Limiting oxygen availability improved the overall performance with regards to total nitrogen (TN) removal by providing a better environment for anaerobic ammonium oxidation (Anammox) while limiting complete nitrification. Oxygen profiles were measured in the iMABR over time at different biofilms depths, showing that intermittent aeration led to various oxygen concentrations and temporal variations in the oxygen availabilities at different depths of the biofilm. Also, N2O emissions from the MABRs differed greatly between the different systems, but still remained lower compared to other reactor configurations for nitrogen removal, making the MABR technology a worthy alternative. The results showed large differences between the operating strategies of the MABRs and can help to gain more insight into the specific properties of MABRs for nitrogen removal.

Quest for Nitrous Oxide-reducing Bacteria Present in an Anammox Biofilm Fed with Nitrous Oxide.

N2O-reducing bacteria have been examined and harnessed to develop technologies that reduce the emission of N2O, a greenhouse gas produced by biological nitrogen removal. Recent investigations using omics and physiological activity approaches have revealed the ecophysiologies of these bacteria during nitrogen removal. Nevertheless, their involvement in| |anammox processes remain unclear. Therefore, the present study investigated the identity, genetic potential, and activity| |of N2O reducers in an anammox reactor. We hypothesized that N2O is limiting for N2O-reducing bacteria| |and an| |exogeneous N2O supply enriches as-yet-uncultured N2O-reducing bacteria. We conducted a 1200-day incubation of N2O-reducing bacteria in an anammox consortium using gas-permeable membrane biofilm reactors (MBfRs), which efficiently supply N2O in a bubbleless form directly to a biofilm grown on a gas-permeable membrane. A 15N tracer test indicated that the supply of N2O resulted in an enriched biomass with a higher N2O sink potential. Quantitative PCR and 16S rRNA amplicon sequencing revealed Clade II nosZ type-carrying N2O-reducing bacteria as protagonists of N2O sinks. Shotgun metagenomics showed the genetic potentials of the predominant Clade II nosZ-carrying bacteria, Anaerolineae and Ignavibacteria in MBfRs. Gemmatimonadota and non-anammox Planctomycetota increased their abundance in MBfRs despite their overall lower abundance. The implication of N2O as an inhibitory compound scavenging vitamin B12, which is essential for the synthesis of methionine, suggested its limited suppressive effect on the growth of B12-dependent bacteria, including N2O reducers. We identified Dehalococcoidia and Clostridia as predominant N2O sinks in an anammox consortium fed exogenous N2O because of the higher metabolic potential of vitamin B12-dependent biosynthesis.

Comprehensive mutation profiling from wastewater in southern Germany extends evidence of circulating SARS-CoV-2 diversity beyond mutations characteristic for Omicron.

Tracking SARS-CoV-2 variants in wastewater is primarily performed by detecting characteristic mutations of the variants. Unlike the Delta variant, the emergence of the Omicron variant and its sublineages as variants of concern has posed a challenge in using characteristic mutations for wastewater surveillance. In this study, we monitored the temporal and spatial variation of SARS-CoV-2 variants by including all the detected mutations and compared whether limiting the analyses to characteristic mutations for variants like Omicron impact the outcomes. We collected 24-hour composite samples from 15 wastewater treatment plants (WWTP) in Hesse and sequenced 164 wastewater samples with a targeted sequencing approach from September 2021 to March 2022. Our results show that comparing the number of all the mutations against the number of the characteristic mutations reveals a different outcome. A different temporal variation was observed for the ORF1a and S gene. As Omicron became dominant, we observed an increase in the overall number of mutations. Based on the characteristic mutations of the SARS-CoV-2 variants, a decreasing trend for the number of ORF1a and S gene mutations was noticed, though the number of known characteristic mutations in both genes is higher in Omicron than Delta.

Quantification of microplastics in wastewater systems of German industrial parks and their wastewater treatment plants.

Microplastics (MP) enter the aquatic environment via several pathways. Many research groups have focused on municipal discharge, while research on industrial sources is rare. This study provides one of the first insights into MP occurrence and distribution in the wastewater systems of industrial parks (IPs) and their wastewater treatment plants (IPWWTPs). The effluents from production plants as well as influent, effluent, and internal samples from the IPWWTPs were assessed. Sampling methods for parallel MP mass and number analyses were developed for varying conditions. The total item emissions of MP (≥10 µm) into the environment were analyzed using µ-Raman spectroscopy and ranged from 3 · 102 to 8 · 104 MP m-3, with a median of 6 · 103 MP m-3 per IPWWTP. Masses analyzed using differential scanning calorimetry showed an MP mass discharge into the environment of 0.2 to 11 mg m-3 with a median of 3.7 mg m-3 per IPWWTP. MP item concentrations within an IPWWTP varied by two to three log levels over several days. Fibers were rare in all samples. Polymer types varied depending on the types of industrial sites and the production plants discharging into the IPWWTP. Within an IP, MP could be allocated to its dischargers, which could be useful for future regulatory requirements. Further research is needed to include different types of IPs producing various polymers and additional processing plants to expand this data set.

Bacterial inactivation processes in water disinfection - mechanistic aspects of primary and secondary oxidants - A critical review.

Water disinfection during drinking water production is one of the most important processes to ensure safe drinking water, which is gaining even more importance due to the increasing impact of climate change. With specific reaction partners, chemical oxidants can form secondary oxidants, which can cause additional damage to bacteria. Cases in point are chlorine dioxide which forms free available chlorine (e.g., in the reaction with phenol) and ozone which can form hydroxyl radicals (e.g., during the reaction with natural organic matter). The present work reviews the complex interplay of all these reactive species which can occur in disinfection processes and their potential to affect disinfection processes. A quantitative overview of their disinfection strength based on inactivation kinetics and typical exposures is provided. By unifying the current data for different oxidants it was observable that cultivated wild strains (e.g., from wastewater treatment plants) are in general more resistant towards chemical oxidants compared to lab-cultivated strains from the same bacterium. Furthermore, it could be shown that for selective strains chlorine dioxide is the strongest disinfectant (highest maximum inactivation), however as a broadband disinfectant ozone showed the highest strength (highest average inactivation). Details in inactivation mechanisms regarding possible target structures and reaction mechanisms are provided. Thereby the formation of secondary oxidants and their role in inactivation of pathogens is decently discussed. Eventually, possible defense responses of bacteria and additional effects which can occur in vivo are discussed.

Effects of scouring on membrane aerated biofilm reactor performance and microbial community composition.

This study investigated the effects of scouring on Membrane Aerated Biofilm Reactors (MABRs). Laboratory-scale MABRs were operated under conditions typical for municipal wastewater. Scouring was induced by bubbling dinitrogen gas through the reactor bulk at low and high frequencies (LF and HF). At low nitrogen surface loads, almost complete ammonium removal was observable while HF scouring resulted in less total nitrogen (TN) removal compared to LF scouring. High nitrogen surface loads combined with HF scouring resulted in a higher TN removal as LF scouring. HF scouring resulted in around four times more sludge production and less residual biofilm mass compared to LF scouring. 16S amplicon sequencing of the biofilm, detached biomass and flocs revealed major differences between the microbial community compositions of these fractions. These results indicate that by varying the scouring strategy is a potential control mechanism for MABR operation and can help to reach specific treatment targets.

Early Detection of SARS-CoV-2 Omicron BA.4 and BA.5 in German Wastewater.

Wastewater-based SARS-CoV-2 epidemiology (WBE) has been established as an important tool to support individual testing strategies. The Omicron sub-variants BA.4/BA.5 have spread globally, displacing the preceding variants. Due to the severe transmissibility and immune escape potential of BA.4/BA.5, early monitoring was required to assess and implement countermeasures in time. In this study, we monitored the prevalence of SARS-CoV-2 BA.4/BA.5 at six municipal wastewater treatment plants (WWTPs) in the Federal State of North Rhine-Westphalia (NRW, Germany) in May and June 2022. Initially, L452R-specific primers/probes originally designed for SARS-CoV-2 Delta detection were validated using inactivated authentic viruses and evaluated for their suitability for detecting BA.4/BA.5. Subsequently, the assay was used for RT-qPCR analysis of RNA purified from wastewater obtained twice a week at six WWTPs. The occurrence of L452R carrying RNA was detected in early May 2022, and the presence of BA.4/BA.5 was confirmed by variant-specific single nucleotide polymorphism PCR (SNP-PCR) targeting E484A/F486V and NGS sequencing. Finally, the mutant fractions were quantitatively monitored by digital PCR, confirming BA.4/BA.5 as the majority variant by 5 June 2022. In conclusion, the successive workflow using RT-qPCR, variant-specific SNP-PCR, and RT-dPCR demonstrates the strength of WBE as a versatile tool to rapidly monitor variants spreading independently of individual test capacities.

Semi-crystalline microplastics in wastewater plant effluents and removal efficiencies of post-treatment filtration systems.

Microplastics (MPs) are ubiquitous in the environment and have been found in every environmental compartment. Wastewater and wastewater treatment plants (WWTPs) have been identified as possible point sources contributing to the emission of microplastic particles (MPP) into the aquatic environment. So far, MPP in wastewater effluents have mainly been analyzed by spectroscopic methods resulting in concentrations as number per volume. In this study, we present mass concentrations in the secondary effluents of four German municipal WWTPs, removal efficiencies of seven post-treatment systems and the resulting load emissions. Differential Scanning Calorimetry (DSC) was used for the analysis of semi-crystalline MPs. The concentrations of secondary effluents ranged from 0.1 to 19.6 µg L-1. Removal efficiencies > 94% were found for a microfiltration membrane (MF), two cloth types of a pile cloth media filter (PCMF), a micro strainer, a discontinuous downflow granulated activated carbon filter (GAC) and a powdered activated carbon (PAC) stage with clarifier and rapid sand filtration. A rapid sand filter (RSF) at WWTP B showed a removal efficiency of 82.38%. Only a continuous upflow GAC filter at WWTP C proved to be unsuitable for MP removal with an average removal efficiency of 1.9%.

Wastewater surveillance allows early detection of SARS-CoV-2 omicron in North Rhine-Westphalia, Germany.

Wastewater-based epidemiology (WBE) has demonstrated its importance to support SARS-CoV-2 epidemiology complementing individual testing strategies. Due to their immune-evasive potential and the resulting significance for public health, close monitoring of SARS-CoV-2 variants of concern (VoC) is required to evaluate the regulation of early local countermeasures. In this study, we demonstrate a rapid workflow for wastewater-based early detection and monitoring of the newly emerging SARS-CoV-2 VoCs Omicron in the end of 2021 at the municipal wastewater treatment plant (WWTP) Emschermuendung (KLEM) in the Federal State of North-Rhine-Westphalia (NRW, Germany). Initially, available primers detecting Omicron-related mutations were rapidly validated in a central laboratory. Subsequently, RT-qPCR analysis of purified SARS-CoV-2 RNA was performed in a decentral PCR laboratory in close proximity to KLEM. This decentralized approach enabled the early detection of K417N present in Omicron in samples collected on 8th December 2021 and the detection of further mutations (N501Y, Δ69/70) in subsequent biweekly sampling campaigns. The presence of Omicron in wastewater was confirmed by next generation sequencing (NGS) in a central laboratory with samples obtained on 14th December 2021. Moreover, the relative increase of the mutant fraction of Omicron was quantitatively monitored over time by dPCR in a central PCR laboratory starting on 12th December 2021 confirming Omicron as the dominant variant by the end of 2021. In conclusions, WBE plays a crucial role in surveillance of SARS-CoV-2 variants and is suitable as an early warning system to identify variant emergence. In particular, the successive workflow using RT-qPCR, RT-dPCR and NGS demonstrates the strength of WBE as a versatile tool to monitor variant spreading.

Are K-strategists yield-strategists in disguise? An example from autotrophic nitrogen removal.

The behavior of heterotrophic bacteria growing in systems with low or no external supply of chemical oxygen demand (COD) has become more relevant within the wastewater context. Growth strategies help to clarify how bacteria behave and adapt to different environmental conditions. In the case of substrate limited conditions, research has been mainly focused on the k-strategy, whereas another important strategy: the yield strategy has not been explored intensely. Some authors have, however, demonstrated the implications of bacteria pursuing the yield strategy when living in structured environments and facing low-substrate concentrations. This study uses a one-dimensional biofilm model to study the influence of the affinity constant, the maximum growth rate, and the growth yield on the heterotrophic formation of dinitrogen gas (N2 ) in a completely autotrophic partial nitritation anammox system. The effect of these parameters on the composition and the diversity of the heterotrophic community is also evaluated. In a first scenario, heterotrophic bacteria are allowed to grow only on the COD produced by biomass decay. In a second step, the competition with a second group of heterotrophs using external COD as electron donor is assessed. For both evaluated scenarios, the results suggest that the yield plays a crucial role in the heterotrophic biomass and dinitrogen gas formation. Moreover, in the case of the community diversity the yield seems to be the decisive parameter. Finally, we conceptually compared the K and the yield strategy and give some insight to the possibility of both either being closely related or even being the same strategy.

Prevalence and circulation patterns of SARS-CoV-2 variants in European sewage mirror clinical data of 54 European cities.

For community-level monitoring, the European Commission under the EU Sewage Sentinel System recommends wastewater-based SARS-CoV-2 surveillance. Tracking SARS-CoV-2 variants in a community is pivotal for appropriate public health response. Genome sequencing of SARS-CoV-2 in wastewater samples for tracking variants is challenging, often resulting in low coverage genome sequences, thereby impeding the detection of the SARS-CoV-2 mutations. Therefore, we aimed at high-coverage SARS-CoV-2 genome sequences from sewage samples which we successfully accomplished. This first pan-European surveillance compared the mutation profiles associated with the variants of concerns: B.1.1.7, P.1, B.1.351 and B.1.617.2 across 20 European countries, including 54 municipalities. The results highlight that SARS-CoV-2 variants detected in the wastewater samples mirror the variants profiles reported in clinical data. This study demonstrated that >98% coverage of SARS-CoV-2 genomic sequences is possible and can be used to track SARS-CoV-2 mutations in wastewater to support identifying variants circulating in a city at the community level.

Genome Sequencing of Wastewater Confirms the Arrival of the SARS-CoV-2 Omicron Variant at Frankfurt Airport but Limited Spread in the City of Frankfurt, Germany, in November 2021.

Wastewater-based severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) surveillance of Frankfurt Airport by genome sequencing was used to detect SARS-CoV-2 variants entering the region. In November 2021, we found all characteristic mutations of Omicron in wastewater originating from Frankfurt Airport before the first confirmed clinical report from an arriving passenger on 26 November 2021.

Monitoring SARS-CoV-2 in sewage: Toward sentinels with analytical accuracy.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemia has been one of the most difficult challenges humankind has recently faced. Wastewater-based epidemiology has emerged as a tool for surveillance and mitigation of potential viral outbreaks, circumventing biases introduced by clinical patient testing. Due to the situation urgency, protocols followed for isolating viral RNA from sewage were not adapted for such sample matrices. In parallel to their implementation for fast collection of data to sustain surveillance and mitigation decisions, molecular protocols need to be harmonized to deliver accurate, reproducible, and comparable analytical outputs. Here we studied analytical variabilities linked to viral RNA isolation methods from sewage. Three different influent wastewater volumes were used to assess the effects of filtered volumes (50, 100 or 500 mL) for capturing viral particles. Three different concentration strategies were tested: electronegative membranes, polyethersulfone membranes, and anion-exchange diethylaminoethyl cellulose columns. To compare the number of viral particles, different RNA isolation methods (column-based vs. magnetic beads) were compared. The effect of extra RNA purification steps and different RT-qPCR strategies (one step vs. two-step) were also evaluated. Results showed that the combination of 500 mL filtration volume through electronegative membranes and without multiple RNA purification steps (using column-based RNA purification) using two-step RT-qPCR avoided false negatives when basal viral load in sewage are present and yielded more consistent results during the surveillance done during the second-wave in Delft (The Hague area, The Netherlands). By paving the way for standardization of methods for the sampling, concentration and molecular detection of SARS-CoV-2 viruses from sewage, these findings can help water and health surveillance authorities to use and trust results coming from wastewater based epidemiology studies in order to anticipate SARS-CoV-2 outbreaks.

Elevated levels of antibiotic resistance in groundwater during treated wastewater irrigation associated with infiltration and accumulation of antibiotic residues.

Treated wastewater irrigation (TWW) releases antibiotics and antibiotic resistance genes (ARGs) into the environment and might thus promote the dissemination of antibiotic resistance in groundwater (GW). We hypothesized that TWW irrigation increases ARG abundance in GW through two potential mechanisms: the contamination of GW with resistant bacteria and the accumulation of antibiotics in GW. To test this, the GW below a real-scale TWW-irrigated field was sampled for six months. Sampling took place before, during and after high-intensity TWW irrigation. Samples were analysed with 16S rRNA amplicon sequencing, qPCR of six ARGs and the class 1 integron-integrase gene intI1, while liquid chromatography tandem mass spectrometry was performed to detect antibiotic and pharmaceutical residues. Absolute abundance of 16S rRNA in GW decreased rather than increased during long-term irrigation. Also, the relative abundance of TWW-related bacteria did not increase in GW during long-term irrigation. In contrast, long-term TWW irrigation increased the relative abundance of sul1 and intI1 in the GW microbiome. Furthermore, GW contained elevated concentrations of sulfonamide antibiotics, especially sulfamethoxazole, to which sul1 confers resistance. Total sulfonamide concentrations in GW correlated with sul1 relative abundance. Consequently, TWW irrigation promoted sul1 and intI1 dissemination in the GW microbiome, most likely due to the accumulation of drug residues.

Combination of 15N Tracer and Microbial Analyses Discloses N2O Sink Potential of the Anammox Community.

Although nitrogen removal by partial nitritation and anammox is more cost-effective than conventional nitrification and denitrification, one downside is the production and accumulation of nitrous oxide (N2O). The potential exploitation of N2O-reducing bacteria, which are resident members of anammox microbial communities, for N2O mitigation would require more knowledge of their ecophysiology. This study investigated the phylogeny of resident N2O-reducing bacteria in an anammox microbial community and quantified individually the processes of N2O production and N2O consumption. An up-flow column-bed anammox reactor, fed with NH4+ and NO2- and devoid of oxygen, emitted N2O at an average conversion ratio (produced N2O: influent nitrogen) of 0.284%. Transcriptionally active and highly abundant nosZ genes in the reactor biomass belonged to the Burkholderiaceae (clade I type) and Chloroflexus genera (clade II type). Meanwhile, less abundant but actively transcribing nosZ strains were detected in the genera Rhodoferax, Azospirillum, Lautropia, and Bdellovibrio and likely act as an N2O sink. A novel 15N tracer method was adapted to individually quantify N2O production and N2O consumption rates. The estimated true N2O production rate and true N2O consumption rate were 3.98 ± 0.15 and 3.03 ± 0.18 mgN·gVSS-1·day-1, respectively. The N2O consumption rate could be increased by 51% (4.57 ± 0.51 mgN·gVSS-1·day-1) with elevated N2O concentrations but kept comparable irrespective of the presence or absence of NO2-. Collectively, the approach allowed the quantification of N2O-reducing activity and the identification of transcriptionally active N2O reducers that may constitute as an N2O sink in anammox-based processes.

Metagenomic Insights Into Functional and Taxonomic Compositions of an Activated Sludge Microbial Community Treating Leachate of a Completed Landfill: A Pathway-Based Analysis.

Upcycling wastes into valuable products by mixed microbial communities has recently received considerable attention. Sustainable production of high-value substances from one-carbon (C1) compounds, e.g., methanol supplemented as an external electron donor in bioreactors for wastewater treatment, is a promising application of upcycling. This study undertook a gene-centric approach to screen valuable production potentials from mixed culture biomass, removing organic carbon and nitrogen from landfill leachate. To this end, the microbial community of the activated sludge from a landfill leachate treatment plant and its metabolic potential for the production of seven valuable products were investigated. The DNA extracted from the activated sludge was subjected to shotgun metagenome sequencing to analyze the microbial taxonomy and functions associated with producing the seven products. The functional analysis confirmed that the activated sludge could produce six of the valuable products, ectoine, polyhydroxybutyrate (PHB), zeaxanthin, astaxanthin, acetoin, and 2,3-butanediol. Quantification of the detected functional gene hit numbers for these valuable products as a primary trial identified a potential rate-limiting metabolic pathway, e.g., conversion of L-2,4-diaminobutyrate into N-γ-acetyl-L2,4,-diaminobutyrate during the ectoine biosynthesis. Overall, this study demonstrated that primary screening by the proposed gene-centric approach can be used to evaluate the potential for the production of valuable products using mixed culture or single microbe in engineered systems. The proposed approach can be expanded to sites where water purification is highly required, but resource recovery, or upcycling has not been implemented.

A multi-component model for granular activated carbon filters combining biofilm and adsorption kinetics.

Along with the rise of biological active granular activated carbon (bGAC) filtration as advanced treatment technology for wastewater treatment plant (WWTP) effluents, the mathematical representation of such systems is gaining increasing importance. This work introduces a model that describes the performance of bGAC-filters for Dissolved Organic Carbon (DOC) removal from a WWTP effluent. The DOC removal within bGAC-filters is accomplished by two mechanisms: adsorptive removal and biological transformation. An appropriate representation of the adsorptive removal requires the DOC to be divided into fictive fractions according to its adsorbability. Likewise, a further DOC classification according to its biodegradability is necessary. Modeling a bGAC-filter then becomes a multi-component adsorption problem, with the simultaneous occurrence of DOC degradation within a biofilm. For dealing with this modeling task, this work integrated the Ideal Adsorbed Solution (IAS) theory into a traditional biofilm model compatible with the Activated Sludge Model (ASM) Framework. For the description of the adsorption dynamics, a Freundlich isotherm for the equilibrium and a pseudo first order model for the kinetics were selected. The biofilm consisted of heterotrophic bacteria able to oxidize DOC using oxygen as electron acceptor. The correctness of the model was evaluated using experimental data from a pilot plant. The predicted DOC breakthrough curve satisfactorily fitted the experimental measurements for empty bed contact times (EBCT) of 6, 12, 24 and 33 min. Moreover, the model predicted the relationship between EBCT, DOC removal and bGAC-filter lifespan. The developed model is the first that combines multi-component adsorption and biofilm kinetics in a wastewater treatment context.

Metatranscriptomic Analysis Reveals SARS-CoV-2 Mutations in Wastewater of the Frankfurt Metropolitan Area in Southern Germany.

We report a sequencing analysis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in wastewater samples collected in the Frankfurt metropolitan area of Germany. The majority of the detected mutations have been identified only in clinical genomes outside Frankfurt, indicating that the sequencing of SARS-CoV-2 RNA in wastewater can provide insights into emerging variants in a city.

Long-term monitoring of SARS-CoV-2 RNA in wastewater of the Frankfurt metropolitan area in Southern Germany.

Wastewater-based epidemiology (WBE) is a great approach that enables us to comprehensively monitor the community to determine the scale and dynamics of infections in a city, particularly in metropolitan cities with a high population density. Therefore, we monitored the time course of the SARS-CoV-2 RNA concentration in raw sewage in the Frankfurt metropolitan area, the European financial center. To determine the SARS-CoV-2 RNA concentration in sewage, we continuously collected 24 h composite samples twice a week from two wastewater treatment plant (WWTP) influents (Niederrad and Sindlingen) serving the Frankfurt metropolitan area and performed RT-qPCR analysis targeting three genes (N gene, S gene, and ORF1ab gene). In August, a resurgence in the SARS-CoV-2 RNA load was observed, reaching 3 × 1013 copies/day, which represented similar levels compared to April with approx. 2 × 1014 copies/day. This corresponds to a continuous increase again in COVID-19 cases in Frankfurt since August, with an average of 28.6 incidences, compared to 28.7 incidences in April. Different temporal dynamics were observed between different sampling points, indicating local dynamics in COVID-19 cases within the Frankfurt metropolitan area. The SARS-CoV-2 RNA load to the WWTP Niederrad ranged from approx. 4 × 1011 to 1 × 1015 copies/day, the load to the WWTP Sindlingen from approx. 1 × 1011 to 2 × 1014 copies/day, which resulted in a preceding increase in these loading in July ahead of the weekly averaged incidences. The study shows that WBE has the potential as an early warning system for SARS-CoV-2 infections and a monitoring system to identify global hotspots of COVID-19.