Wastewater Treatment Plants In Japan Research Articles

High-throughput microfluidic quantitative PCR system for the simultaneous detection of antibiotic resistance genes and bacterial and viral pathogens in wastewater

Comprehensive data on bacterial and viral pathogens of diarrhea and studies applying culture-independent methods for examining antibiotic resistance in wastewater are lacking. This study aimed to simultaneously quantify antibiotic resistance genes (ARGs), class 1 integron-integrase (int1), bacterial and viral pathogens of diarrhea, 16S rRNA, and other indicators using a high-throughput quantitative PCR (HT-qPCR) system. Thirty-six grab wastewater samples from a wastewater treatment plant in Japan, collected three times a month between August 2022 and July 2023, were centrifuged, followed by nucleic acid extraction, reverse transcription, and HT-qPCR. Fourteen targets were included, and HT-qPCR was performed on the Biomark X9™ System (Standard BioTools). For all qPCR assays, R2 was ≥0.978 and the efficiencies ranged from 90.5% to 117.7%, exhibiting high performance. Of the 36 samples, 20 (56%) were positive for Norovirus genogroup II (NoV-GII), whereas Salmonella spp. and Campylobacter jejuni were detected in 24 (67%) and Campylobacter coli in 13 (36%) samples, with mean concentrations ranging from 3.2 ± 0.8 to 4.7 ± 0.3 log10 copies/L. NoV-GII detection ratios and concentrations were higher in winter and spring. None of the pathogens of diarrhea correlated with acute gastroenteritis cases, except for NoV-GII, suggesting the need for data on specific bacterial infections to validate bacterial wastewater-based epidemiology (WBE). All samples tested positive for sul1, int1, and blaCTX-M, irrespective of season. The less explored blaNDM-1 showed a wide prevalence (>83%) and consistent abundance ranging from 4.3 ± 1.0 to 4.9 ± 0.2 log10 copies/L in all seasons. sul1 was the predominant ARG, whereas absolute abundances of 16S rRNA, int1, and blaCTX-M varied seasonally. int1 was significantly correlated with blaCTX-M in autumn and spring, whereas it showed no correlation with blaNDM-1, questioning the applicability of int1 as a sole indicator of overall resistance determinants. This study exhibited that the HT-qPCR system is pivotal for WBE.

Group A Streptococcus pyogenes in wastewater: Applicability of wastewater-based epidemiology for monitoring the prevalence of GAS pharyngitis during the late COVID-19 pandemic phase

Streptococcus pyogenes, Group A Streptococcus (GAS), is a human pathogen that causes a spectrum of diseases from mild to severe, including GAS pharyngitis, a common acute respiratory disease in developed countries. Although wastewater-based epidemiology (WBE) has been extensively used to monitor viral pathogens such as severe acute respiratory syndrome coronavirus 2, its applicability to S. pyogenes remains unexplored. This study was conducted to investigate the feasibility of detecting and quantifying S. pyogenes in wastewater by quantitative polymerase chain reaction (qPCR) and evaluate the applicability of WBE for monitoring the prevalence of GAS pharyngitis. A total of 52 grab influent samples were collected from a wastewater treatment plant in Japan once a week between March 2023 and February 2024. The samples were centrifuged, followed by nucleic acid extraction and qPCR for the S. pyogenes-specific genes speB and spy1258. Of the 52 samples, 90 % and 81 % were positive for speB and spy1258 genes, respectively, indicating the feasibility of S. pyogenes for wastewater surveillance. However, the percentage of quantifiable samples for speB gene was significantly higher in winter than in spring and summer. Similarly, the concentrations of both genes in wastewater samples were significantly higher in winter (speB, 4.1 ± 0.27 log10 copies/L; spy1258, 4.1 ± 0.28 log10 copies/L; One-way ANOVA, p < 0.01) than in spring and summer. Higher concentrations and detection ratios of S. pyogenes genes were observed during increased GAS pharyngitis cases in the catchment. Significant moderate correlations were observed between target gene concentrations and reported GAS pharyngitis cases. This study enhances the understanding role of WBE in monitoring and managing infectious diseases within communities.

Optimization of the 5-plex digital PCR workflow for simultaneous monitoring of SARS-CoV-2 and other pathogenic viruses in wastewater

Wastewater-based epidemiology is a valuable tool for monitoring pathogenic viruses in the environment, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19). While quantitative polymerase chain reaction (qPCR) is widely used for pathogen surveillance in wastewater, it can be affected by inhibition and is limited to relative quantification. Digital PCR (dPCR) offers potential solutions to these limitations. In this study, a 5-plex dPCR workflow was optimized for the simultaneous detection of SARS-CoV-2, influenza A virus, enteroviruses (EnV), and noroviruses of genogroups I (NoV-GI) and GII (NoV-GII) in wastewater samples. Wastewater samples (n = 36) were collected from a wastewater treatment plant in Japan between August and October 2022. The optimization included the evaluation of singleplex and 5-plex dPCR assays, and two different concentration methods, extraction kits, and dPCR approaches. The performance of singleplex and 5-plex dPCR assays showed comparable linearity and reliability, with the 5-plex assays showing greater efficiency. The polyethylene glycol (PEG) precipitation method showed better performance over the centrifugation method, two-step reverse transcription (RT)-dPCR over the one-step RT-dPCR, and AllPrep PowerViral DNA/RNA Kit showed better performance than the QIAamp Viral RNA Mini Kit. The optimal workflow therefore included PEG precipitation, the AllPrep PowerViral DNA/RNA Kit, and two-step RT-dPCR. This workflow was selected to monitor the presence of SARS-CoV-2 and other pathogenic viruses in wastewater samples in a 5-plex dPCR approach, yielding promising results. SARS-CoV-2 RNA was detected in the majority of samples, with NoV-GI, NoV-GII, and EnV also being detected. The successful optimization and application of the 5-plex dPCR assay for pathogen surveillance in wastewater offers significant benefits, including enhanced community health assessment and more effective responses to public health threats.

Development of highly sensitive one-step reverse transcription-quantitative PCR for SARS-CoV-2 detection in wastewater

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants is a major public health concern that has highlighted the need to monitor circulating strains to better understand the coronavirus disease 2019 (COVID-19) pandemic. This study was carried out to monitor SARS-CoV-2 RNA and its variant-specific mutations in wastewater using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). One-step RT-qPCR using the SARS-CoV-2 Detection RT-qPCR Kit for Wastewater (Takara Bio), which amplified two N-gene regions simultaneously using CDC N1 and N2 assays with a single fluorescence dye, demonstrated better performance in detecting SARS-CoV-2 RNA (positive ratio, 66 %) compared to two-step RT-qPCR using CDC N1 or N2 assay (40 % each, and 52 % when combined), with significantly lower Ct values. The one-step RT-qPCR assay detected SARS-CoV-2 RNA in 59 % (38/64) of influent samples collected from a wastewater treatment plant in Japan between January 2021 and March 2022. The correlation between the concentration of SARS-CoV-2 RNA in the wastewater and the number of COVID-19 cases reported each day for 7 days pre- and post-sampling was significant (p < 0.05, r = 0.76 ± 0.03). Thirty-one influent samples which showed two-well positive for SARS-CoV-2 RNA were further tested by six mutations site-specific one-step RT-qPCR (E484K, L452R, N501Y, T478K, G339D, and E484A mutations). The N501Y mutation was detected between March and June 2021 but was replaced by the L452R and T478K mutations between July and October 2021, reflecting the shift from Alpha to Delta variants in the study region. The G339D and E484A mutations were identified in January 2022 and later when the incidence of the Omicron variant peaked. These findings indicate that wastewater-based epidemiology has the epidemiological potential to complement clinical tests to track the spread of COVID-19 and monitor variants circulating in communities.

Concurrent Analysis of 84 Compounds among Emerging Contaminants Listed by the Ministry of the Environment, Japan, in Domestic Wastewater Treatment Plants Using Liquid Chromatography and High-resolution Mass Spectrometry (LC-HRMS)

The Ministry of the Environment, Japan has listed 207 items of emerging contaminants that are to be surveyed (“Items to be surveyed”, Youchousa-koumoku) to study the contamination in the water environment. Wastewater treatment plants (WWTPs) are one of the main sources of these contaminants in the water environment. In the current study, 84 compounds in the list of “Items to be surveyed” were concurrently analyzed in samples from 10 different WWTPs in Japan using liquid chromatography and high-resolution mass spectrometry. Samples were collected at three stages; i) primary effluent, ii) secondary effluent, and iii) final effluent. Impact of solid phase extraction and wastewater matrix on the recovery of the compounds was evaluated. 29 compounds were detected in at least 4 WWTPs. Triethanolamine, N,N-dimethyldodecylamine, 2-(2-ethoxyethoxy) ethanol, diethanolamine, and N,N-dimethyalacetamide had high removal (> 60%) in at least 6 treatment plants. The conventional activated sludge process was found to have lower removal efficiency for some compounds than the other processes. Decanoic acid had a concentration higher than its predicted no effect concentration (PNEC) and other three compounds had concentrations greater than PNEC/10 in final effluent.

Survey of elemental composition in dewatered sludge in Japan

The elemental composition of 120 dewatered sludge samples from 32 wastewater treatment plants in Japan was analyzed, and the influential factors determining sludge elemental composition were clarified. Through Hayashi's quantification method I, the relationship and influence of each element to the dominant categories were confirmed, and the correlation between the elements was also analyzed. The Van Krevelen diagram was used to determine the similarity in organic contents between sludge and biomass/fossil fuels. Principal component and cluster analyses were used to verify the identification of influential factors. Finally, a standard composition of dewatered sludge in Japan was defined based on different sewage collection systems and digestion processes, shown to have the greatest influence on sludge composition. The potential of sludge for agricultural and cement industrial recycling was also assessed.

Development of a Portable Detection Method for Enteric Viruses from Ambient Air and Its Application to a Wastewater Treatment Plant.

The ambient air from wastewater treatment plants has been considered as a potential source of pathogenic microorganisms to cause an occupational risk for the workers of the plants. Existing detection methods for enteric viruses from the air using a liquid as the collection medium therefore require special care to handle on-site. Knowledge accumulation on airborne virus risks from wastewater has been hindered by a lack of portable and handy collection methods. Enteric viruses are prevalent at high concentrations in wastewater; thus, the surrounding air may also be a potential source of viral transmission. We developed a portable collection and detection method for enteric viruses from ambient air and applied it to an actual wastewater treatment plant in Japan. Materials of the collection medium and eluting methods were optimized for real-time polymerase chain reaction-based virus quantification. The method uses a 4 L/min active air sampler, which is capable of testing 0.7–1.6 m3 air after 3–7 h sampling with a detection limit of 102 copies/m3 air in the field. Among 16 samples collected at five to seven locations in three sampling trials (November 2007–January 2008), 56% (9/16) samples were positive for norovirus (NV) GII, with the highest concentration of 3.2 × 103 copies/m3 air observed at the sampling point near a grit chamber. Adenoviruses (4/16), NV GI (6/16), FRNA bacteriophages GIII (3/16), and enteroviruses (3/16) were also detected but at lower concentrations. The virus concentration in the air was associated with that of the wastewater at each process. The results imply that the air from the sewer pipes or treatment process is contaminated by enteric viruses and thus special attention is needed to avoid accidental ingestion of viruses via air.

Characterization of NDM-5- and CTX-M-55-coproducing Escherichia coli GSH8M-2 isolated from the effluent of a wastewater treatment plant in Tokyo Bay.

PurposeNew Delhi metallo-β-lactamase (NDM)-5-producing Enterobacteriaceae have been detected in rivers, sewage, and effluents from wastewater treatment plants (WWTPs). Environmental contamination due to discharged effluents is of particular concern as NDM variants may be released into waterways, thereby posing a risk to humans. In this study, we collected effluent samples from a WWTP discharged into a canal in Tokyo Bay, Japan.MethodsTesting included the complete genome sequencing of Escherichia coli GSH8M-2 isolated from the effluent as well as a gene network analysis.ResultsThe complete genome sequencing of GSH8M-2 revealed that it was an NDM-5-producing E. coli strain sequence type ST542, which carries multiple antimicrobial resistance genes for β-lactams, quinolone, tetracycline, trimethoprim-sulfamethoxazole, florfenicol/chloramphenicol, kanamycin, and fosfomycin. The blaNDM-5 gene was found in the IncX3 replicon plasmid pGSH8M-2-4. Gene network analysis using 142 IncX3 plasmid sequences suggested that pGSH8M-2-4 is related to both clinical isolates of E. coli and Klebsiella species in Eastern Asia. GSH8M-2 also carries the blaCTX-M-55 gene in IncX1 plasmid pGSH8M-2-3.ConclusionThis is the first report of environmental NDM-5-producing E. coli isolated from a WWTP in Japan. NDM-5 detection is markedly increasing in veterinary and clinical settings, suggesting that dual β-lactamases, such as NDM-5 and CTX-M-55, might be acquired through multiple steps in environment settings. Environmental contamination through WWTP effluents that contain producers of NDM variants could be an emerging potential health hazard. Thus, regular monitoring of WWTP effluents is important for the detection of antimicrobial-resistant bacteria that may be released into the waterways and nearby communities.

Potential KPC-2 carbapenemase reservoir of environmental Aeromonas hydrophila and Aeromonas caviae isolates from the effluent of an urban wastewater treatment plant in Japan.

Summary Aeromonas hydrophila and Aeromonas caviae adapt to saline water environments and are the most predominant Aeromonas species isolated from estuaries. Here, we isolated antimicrobial‐resistant (AMR) Aeromonas strains (A. hydrophila GSH8‐2 and A. caviae GSH8M‐1) carrying the carabapenemase bla KPC‐2 gene from a wastewater treatment plant (WWTP) effluent in Tokyo Bay (Japan) and determined their complete genome sequences. GSH8‐2 and GSH8M‐1 were classified as newly assigned sequence types ST558 and ST13, suggesting no supportive evidence of clonal dissemination. The strains appear to have acquired bla KPC‐2‐positive IncP‐6‐relative plasmids (pGSH8‐2 and pGSH8M‐1‐2) that share a common backbone with plasmids in Aeromonas sp. ASNIH3 isolated from hospital wastewater in the United States, A. hydrophila WCHAH045096 isolated from sewage in China, other clinical isolates (Klebsiella, Enterobacter and Escherichia coli), and wastewater isolates (Citrobacter, Pseudomonas and other Aeromonas spp.). In addition to bla KPC‐2, pGSH8M‐1‐2 carries an IS26‐mediated composite transposon including a macrolide resistance gene, mph(A). Although Aeromonas species are opportunistic pathogens, they could serve as potential environmental reservoir bacteria for carbapenemase and AMR genes. AMR monitoring from WWTP effluents will contribute to the detection of ongoing AMR dissemination in the environment and might provide an early warning of potential dissemination in clinical settings and communities.

Diversity of N-acyl homoserine lactones in activated sludge detected by Fourier transform mass spectrometry

Quorum sensing is recently considered to play important roles in biological wastewater treatment processes. This study aimed to investigate diversity of N-acyl homoserine lactones (AHL) among biological wastewater treatment processes and their locations. We analyzed and quantified 10 AHL in activated sludge at wastewater treatment plants (WWTPs) by Fourier transform mass spectrometry (FTMS) and by bioassay on thin-layer chromatography (TLC). Sludge samples were taken at seven WWTPs in Japan and in Thailand, which were chosen to compare different process types including a pilot-scale membrane bioreactor process. Various AHL were detected in activated sludge, suggesting the presence of quorum sensing activity in many practical WWTPs. Detection of AHL by LC-FTMS was much more sensitive than by TLC bioassay, because TLC bioassay was implied to be affected by the presence of quorum quenching molecules in activated sludge. With LC-FTMS analysis, AHL, which has short straight chain in the acyl group, were more abundant. Especially, C4-HSL, C7-HSL, and C8-HSL were detected widely independent of locations and process types. AO processes had higher AHL productivity than other processes.

Developing of Performance Testing Scheme for Decentralized Domestic Wastewater Treatment Plant in Indonesia

Most of the urban and rural areas in Indonesia still use individual scale of decentralized on-site domestic wastewater treatment such as pit latrines and septic tank. For some building and small community using communal scale of wastewater treatment plant such as fabricated biofilter tank, UASB (up-flow anaerobic sludge blanket), Rotating Biological Contactor (RBC) and wetland. Many application of the decentralized Wastewater Treatment Plants (WWTP) have low performance and the effluent does not meet standard effluent. To ensure the compliance to the regulation, it is necessary to develop the performance testing scheme for decentralized domestic wastewater treatment plant. The objective of the study is to review performance testing scheme in Indonesia, include the legal aspects, existing performance testing procedure for inspection and certification. The target is to develop performance testing schemes consists of testing standard and institutions scheme. The methodology of research are primary and secondary data collection done through field observation and discussion in stakeholder meeting with stakeholder related. The data analysis of this study are comparative study of certification system of decentralized WWTP in Japan, Thailand and Indonesia, descriptive analysis of performance testing method and the role of stakeholders. In conclusion Indonesia has not have regulation regarding certification schemes for decentralized WWTP. The certification schemes is needed to guarantee the WWTP product compliance with the performance standard and effluent standard. The developing decentralized WWTP need the regulation regarding testing body, the provision of performance testing standard, competent testing body, equipment and human resources.

Investigation of detection methods for quantification of Escherichia coli in the wastewater samples and occurrence of E.coli and total coliforms in 54 wastewater treatment plants in Japan

Investigation of detection methods for quantification of <i>Escherichia coli</i> in the wastewater samples and occurrence of <i>E.coli</i> and total coliforms in 54 wastewater treatment plants in Japan

Cake layer bacterial communities during different biofouling stages in full-scale membrane bioreactors

A detailed understanding of the bacterial communities in the cake layers formed on the membrane surface is required to control biofouling in a membrane bioreactor (MBR). This study aimed to investigate the dynamics of the cake layer bacterial communities in full-scale MBRs operated in a wastewater treatment plant in Japan and to identify the key bacteria responsible for cake layer formation. The bacterial communities in the cake layer and the activated sludge were analyzed using 16S rRNA gene amplicon sequencing when biofouling occurred under different fouling conditions. The most dominant phyla in activated sludge were almost always Proteobacteria and Bacteroidetes. By contrast, when the cake layer had unique bacterial communities distinguishable from those in the activated sludge, members of Firmicutes were highly dominant in the cake layer, irrespective of the fouling conditions. This study reported for the first time that Firmicutes play an important role throughout the biofouling process.

Improvement of virus removal by pilot-scale coagulation-ultrafiltration process for wastewater reclamation: Effect of optimization of pH in secondary effluent

Reclaimed water (i.e., reused advanced-treated wastewater) offers an alternative water resource. To reduce the health risks associated with its use, efficient virus removal such as with advanced wastewater treatment processes is important. Virus removal by coagulation followed by ultrafiltration (UF) for the treatment of drinking water has been well examined. But its efficacy in wastewater reclamation purpose using secondary treated effluent (SE) from wastewater treatment plant (WWTP) as feed water is unclear. Here, we optimized the virus removal efficiency of coagulation-UF in pilot-scale wastewater reclamation plants using SE as feed water, using the F-specific RNA bacteriophage MS2 as a model virus, at two wastewater treatment plants in Japan. We investigated how using coagulation as a pretreatment for UF improved virus removal efficiency. The efficiency varied greatly between SEs. To reveal the cause of the variation, we conducted laboratory-scale batch coagulation experiments. The efficiency of viral coagulation was negatively correlated with the concentration of dissolved organic matter in the feed water. The optimum pH for coagulation differed between SEs, and the efficiency of coagulation could be dramatically improved by optimizing the pH. We confirmed that the virus removal efficiency in the pilot-scale facility actually could be improved by adjusting the pH. In addition, we confirmed that coagulation–sedimentation-UF with pH adjustment could operate stably for more than 30 days at the pilot scale, with a high virus removal rate. Thus, the wastewater reclamation process described here offers promise in terms of reduced health risks and practical operation.

Distinct behaviors of infectious F-specific RNA coliphage genogroups at a wastewater treatment plant

The present study aimed to determine the differences in the behaviors of four F-specific RNA (F-RNA) coliphage genogroups (GI–GIV) during wastewater treatment. Raw sewage, aeration tank effluent, secondary-treated sewage, and return activated sludge were collected from a wastewater treatment plant in Japan at monthly intervals between March and December 2011 (n=10 each). F-specific coliphages were detected by plaque assay in all tested samples, with a concentration ranging from −0.10 to 3.66log10plaque-forming units/ml. Subsequently, eight plaques were isolated from each sample, followed by genogroup-specific reverse-transcription quantitative PCR (qPCR) for F-RNA coliphages and qPCR for F-specific DNA (F-DNA) coliphages. GI F-RNA coliphages were the most abundant in the secondary-treated sewage samples (73% of the plaque isolates), while GII F-RNA coliphages were the most abundant in the other three sample types (41–81%, depending on sample type). Based on the results of the quantification and genotyping, the annual mean concentrations of each F-specific coliphage type were calculated, and their reduction ratios during wastewater treatment were compared with those of indicator bacteria (total coliforms and Escherichia coli) and enteric viruses (human adenoviruses and GI and GII noroviruses). The mean reduction ratio of GI F-RNA coliphages was the lowest (0.93 log10), followed by those of the indicator bacteria and enteric viruses (1.59–2.43 log10), GII–GIV F-RNA coliphages (>2.60–3.21 log10), and F-DNA coliphages (>3.41 log10). These results suggest that GI F-RNA coliphages may be used as an appropriate indicator of virus reduction during wastewater treatment.

Occurrence and fate of N-nitrosamines and their formation potential in three wastewater treatment plants in Japan

N-nitrosamines are well known as carcinogens present in the environment. However, studies of the occurrence and fate of N-nitrosamines and their N-nitrosamine formation potential (FP) in wastewater treatment plants (WWTPs) are lacking. Therefore, the objectives of this study were to determine the occurrence of N-nitrosamines in WWTPs, the FP of N-nitrosamines on chloramination during wastewater treatment, and the efficiency of FPNH2Cl reduction by biological treatment. Also, the residual FPNH2Cl in the final discharge was investigated. The efficiencies of removal of N-nitrosamines ranged from 35 to 94% (WWTP O; residential area), from 58 to 98% (WWTP E; industrial area), and from 58% to >99% (WWTP N; industrial area). In WWTP O, the rates of production of N-nitrosodimethylamine (NDMA) (42%) and NDBA (58%) were the highest. In WWTP E, only NDBA (34%) was produced. In WWTP N, NPYR and NPIP (both >99%) were produced. NDMA FPNH2Cl values were very high in influent in all WWTPs. The efficiencies of removal of N-nitrosamine FPNH2Cl during biological treatment ranged from 20% to >99%, but there is no obvious explanation for this variability. Residual N-nitrosamine FPNH2Cl ranged from 2 to 22 ng L(-1). Thus, N-nitrosamines could be produced in water purification or reclamation plants using discharge from WWTPs.

Real-time PCR for rapidly detecting aniline-degrading bacteria in activated sludge

We developed a detection method that uses quantitative real-time PCR (qPCR) and the TaqMan system to easily and rapidly assess the population of aniline-degrading bacteria in activated sludge prior to conducting a biodegradability test on a chemical compound. A primer and probe set for qPCR was designed by a multiple alignment of conserved amino acid sequences encoding the large (α) subunit of aniline dioxygenase. PCR amplification tests showed that the designed primer and probe set targeted aniline-degrading strains such as Acidovorax sp., Gordonia sp., Rhodococcus sp., and Pseudomonas putida, thereby suggesting that the developed method can detect a wide variety of aniline-degrading bacteria. There was a strong correlation between the relative copy number of the α-aniline dioxygenase gene in activated sludge obtained with the developed qPCR method and the number of aniline-degrading bacteria measured by the Most Probable Number method, which is the conventional method, and a good correlation with the lag time of the BOD curve for aniline degradation produced by the biodegradability test in activated sludge samples collected from eight different wastewater treatment plants in Japan. The developed method will be valuable for the rapid and accurate evaluation of the activity of inocula prior to conducting a ready biodegradability test.

Occurrence and reduction of human viruses, F-specific RNA coliphage genogroups and microbial indicators at a full-scale wastewater treatment plant in Japan

To evaluate and compare the reductions of human viruses and F-specific coliphages in a full-scale wastewater treatment plant based on the quantitative PCR (qPCR) and plate count assays. A total of 24 water samples were collected from four locations at the plant, and the relative abundance of human viruses and F-RNA phage genogroups were determined by qPCR. Of the 10 types of viruses tested, enteric adenoviruses were the most prevalent in both influent and effluent wastewater samples. Of the different treatment steps, the activated sludge process was most effective in reducing the microbial loads. Viruses and F-RNA phages showed variable reduction; among them, GI and GIII F-RNA phages showed the lowest and the highest reduction, respectively. Ten types of viruses were present in wastewater that is discharged into public water bodies after treatment. The variability in reduction for the different virus types demonstrates that selection of adequate viral indicators is important for evaluating the efficacy of wastewater treatment and ensuring the water safety. Our comprehensive analyses of the occurrence and reduction of viruses and indicators can contribute to the future establishment of appropriate viral indicators to evaluate the efficacy of wastewater treatment.

Prevalence and Genetic Diversity of Klassevirus in Wastewater in Japan

Klassevirus is a novel virus belonging to the family Picornaviridae. This study examined the prevalence and genetic diversity of klassevirus in wastewater. Raw sewage (100 ml) and secondary-treated sewage (2 l) were collected monthly for 14 months between January 2011 and February 2012 from a wastewater treatment plant in Japan. Klassevirus in the sample was concentrated by the electronegative membrane-vortex method, followed by qualitative detection by means of three types of reverse transcription (RT)-nested polymerase chain reactions (PCRs). Klassevirus was detected in seven of the 14 raw sewage (50 %) and four of the 14 secondary-treated sewage (29 %) samples by the RT-nested PCRs targeting the 2C and/or 3D regions. In contrast, none of the samples tested positive for the virus by the RT-nested PCR targeting the VP0/VP3 region. Based on direct nucleotide sequence analysis of the klassevirus-positive nested PCR fragments, the tested samples showed high nucleotide sequence similarities of 94.7-100.0 % and 93.2-100.0 % in the 2C and 3D regions, respectively, indicating the presence of a single klassevirus strain. To our knowledge, this is the first study evaluating seasonal prevalence and genetic diversity of klassevirus in environmental waters.

Molecular detection and genotyping of human noroviruses in influent and effluent water at a wastewater treatment plant in Japan

To investigate the prevalence, seasonality and genotype distribution of human noroviruses (NoVs) in wastewater in Japan. Influent and effluent water samples were collected monthly for a year from a wastewater treatment plant and examined for the presence of genogroups I and II (GI and GII) NoVs. Using real-time reverse transcription (RT)-PCR assays, 12 (100%) influent and six (50%) effluent samples were positive for both GI and GII NoV genomes, with a higher prevalence in winter. A total of 152 different NoV strains, comprising 84 GI and 68 GII strains, were identified using seminested RT-PCR assays followed by cloning and sequence analysis. These strains were classified into nine GI genotypes (GI/1, 2, 4, 5, 8, 9, 11, 12 and 14) and 13 GII genotypes (GII/1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 15 and 16), showing considerable genetic diversity. Based on the partial capsid gene sequences, we identified a great number of NoV strains belonging to many genotypes, demonstrating that genetically diverse NoV strains are co-circulating in aquatic environments and human populations. Our results clearly demonstrate the seasonal trend and genetic diversity of NoVs in wastewater.