The reproducibility and sensitivity of 36 methods for quantifying the genetic signal of SARS-CoV-2 in wastewater was evaluated in a nationwide interlaboratory assessment in the U.S.

In response to COVID-19, the international water community rapidly developed methods to quantify the SARS-CoV-2 genetic signal in untreated wastewater. Wastewater surveillance using such methods has the potential to complement clinical testing in assessing community health. This interlaboratory assessment evaluated the reproducibility and sensitivity of 36 standard operating procedures (SOPs), divided into eight method groups based on sample concentration approach and whether solids were removed. Two raw wastewater samples were collected in August 2020, amended with a matrix spike (betacoronavirus OC43), and distributed to 32 laboratories across the U.S. Replicate samples analyzed in accordance with the project's quality assurance plan showed high reproducibility across the 36 SOPs: 80% of the recovery-corrected results fell within a band of +/- 1.15-log10 genome copies/L with higher reproducibility observed within a single SOP (standard deviation of 0.13-log10). The inclusion of a solids removal step and the selection of a concentration method did not show a clear, systematic impact on the recovery-corrected results. Other methodological variations (e.g., pasteurization, primer set selection, and use of RT-qPCR or RT-dPCR platforms) generally resulted in small differences compared to other sources of variability. These findings suggest that a variety of methods are capable of producing reproducible results, though the same SOP or laboratory should be selected to track SARS-CoV-2 trends at a given facility. The methods showed a 7-log10 range of recovery efficiency and limit of detection highlighting the importance of recovery correction and the need to consider method sensitivity when selecting methods for wastewater surveillance.

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Effective and sensitive monitoring of human pathogenic bacteria in municipal wastewater treatment is important not only for managing public health risk related to treated wastewater reuse, but also for ensuring proper functioning of the treatment plant. In this study, three different 16S rRNA gene molecular analysis methodologies were employed to screen bacterial pathogens in samples collected at three different stages of an activated sludge plant. Overall bacterial diversity was analyzed using next generation sequencing (NGS) on the Illumina MiSeq platform, as well as PCR-DGGE followed by band sequencing. In addition, a microdiversity analysis was conducted using PCR-DGGE, targeting Escherichia coli. Bioinformatics analysis was performed using QIIME protocol by clustering sequences against the Human Pathogenic Bacteria Database. NGS data were also clustered against the Greengenes database for a genera-level diversity analysis. NGS proved to be the most effective approach screening the sequences of 21 potential human bacterial pathogens, while the E. coli microdiversity analysis yielded one (O157:H7 str. EDL933) out of the two E. coli strains picked up by NGS. Overall diversity using PCR-DGGE did not yield any pathogenic sequence matches even though a number of sequences matched the NGS results. Overall, sequences of Gram-negative pathogens decreased in relative abundance along the treatment train while those of Gram-positive pathogens increased.

Anaerobic membrane bioreactors (AnMBRs) are in use at the full-scale for energy recovery from food waste (FW). In this study, the potential for two-phase (acid/gas) AnMBR treatment of FW was investigated as a strategy to increase microbial diversity, thereby improving performance. Two bench-scale AnMBRs were operated in single-phase (SP) and two-phase (TP) mode across incremental increases in organic loading rate (OLR) from 2.5 to 15 g total chemical oxygen demand (COD) L•d −1 . The TP acid-phase (TP-AP) enriched total VFAs by 3-fold compared to influent FW and harbored a distinct microbial community enriched in fermenters that thrived in the low pH environment. The TP methane phase (TP-MP) showed increased methane production and resilience relative to SP as OLR increased from 3.5 to 10 g COD L•d −1 . SP showed signs of inhibition (i.e., rapid decrease in methane production per OLR) at 10 g COD L•d −1 , whereas both systems were inhibited at 15 g COD L•d −1 . At 10 g COD L• d −1 , where the highest difference in performance was observed (20.3% increase in methane production), activity of syntrophic bacteria in TP-MP was double that of SP. Our results indicate that AnMBRs in TP mode could effectively treat FW at OLRs up to 10 g COD•L day −1 by improving hydrolysis rates, microbial diversity, and syntroph activity, and enriching resistant communities to high OLRs relative to AnMBRs in SP mode.

With accumulating evidence of pulmonary infection via aerosolized nontuberculous mycobacteria (NTM), it is important to characterize their persistence in wastewater treatment, especially in arid regions where treated municipal wastewater is extensively reused. To achieve this goal, microbial diversity of the genus Mycobacterium was screened for clinically and environmentally relevant species using pyrosequencing. Analysis of the postdisinfected treated wastewater showed the presence of clinically relevant slow growers like M. kansasii, M. szulgai, M. gordonae, and M. asiaticum; however, in these samples, rapid growers like M. mageritense occurred at much higher relative abundance. M. asiaticum and M. mageritense have been isolated in pulmonary samples from NTM-infected patients in the region. Diversity analysis along the treatment train found environmentally relevant organisms like M. poriferae and M. insubricum to increase in relative abundance across the chlorine disinfection step. A comparison to qPCR results across the chlorine disinfection step saw no significant change in slow grower counts at CT disinfection values ≤90 mg·min/L; only an increase to 180 mg·min/L in late May brought slow growers to below detection levels. The study confirms the occurrence of clinically and environmentally relevant mycobacteria in treated municipal wastewater, suggesting the need for vigilant monitoring of treated wastewater quality and disinfection effectiveness prior to reuse.

Developing reliable quantitative microbial risk assessment (QMRA) procedures aids in setting recommendations on reuse applications of treated wastewater. In this study, a probabilistic QMRA to determine the risk of Salmonella infections resulting from the consumption of edible crops irrigated with treated wastewater was conducted. Quantitative polymerase chain reaction (qPCR) was used to enumerate Salmonella spp. in post-disinfected samples, where they showed concentrations ranging from 90 to 1,600 cells/100 mL. The results were used to construct probabilistic exposure models for the raw consumption of three vegetables (lettuce, cabbage, and cucumber) irrigated with treated wastewater, and to estimate the disease burden using Monte Carlo analysis. The results showed elevated median disease burden, when compared with acceptable disease burden set by the World Health Organization, which is 10⁻⁶ disability-adjusted life years per person per year. Of the three vegetables considered, lettuce showed the highest risk of infection in all scenarios considered, while cucumber showed the lowest risk. The results of the Salmonella concentration obtained with qPCR were compared with the results of Escherichia coli concentration for samples taken on the same sampling dates.