Next-generation sequencing for surveillance of antimicrobial resistance and pathogenicity in municipal wastewater treatment plants.

The World Health Organization (WHO) ranks antimicrobial resistance (AMR) and various pathogens among the top 10 health threats. It is estimated that by 2050, the number of human deaths due to AMR will reach 10 million annually. On the other hand, several infectious outbreaks such as SARS, H1N1 influenza, Ebola, Zika fever, and COVID-19 have severely affected human populations worldwide in the last 20 years. These recent global diseases have generated the need to monitor outbreaks of pathogens and AMR to establish effective public health strategies. This review presents AMR and pathogenicity associated with wastewater treatment plants (WWTP), focusing on Next Generation Sequencing (NGS) monitoring as a complementary system to clinical surveillance. In this regard, WWTP may be monitored at three main points. First, at the inlet (raw wastewater or influent) to identify a broad spectrum of AMR and pathogens contained in the excretions of residents served by sewer networks, with a specific spatio-temporal location. Second, at the effluent, to ensure the elimination of AMR and pathogens in the treated water, considering the rising demand for safe wastewater reuse. Third, in sewage sludge or biosolids, since their beneficial use or final disposal can represent a significant risk to public health. This review is divided into two sections to address the importance and implications of AMR and pathogen surveillance in wastewater and WWTP, based on NGS. The first section presents the fundamentals of surveillance techniques applied in WWTP (metataxonomics, metagenomics, functional metagenomics, metaviromics, and metatranscriptomics). Their scope and limitations are analyzed to show how microbiological and qPCR techniques complement NGS surveillance, overcoming its limitations. The second section discusses the contribution of 36 NGS research papers on WWTP surveillance, highlighting the current situation and perspectives. In both sections, research challenges and opportunities are presented.

Reuse of Sludge as Organic Soil Amendment: Insights into the Current Situation and Potential Challenges

Sludge generation as an organic by-product of wastewater treatment has seen a consistent increase worldwide due to population growth and industrial activities. This poses a chronic challenge regarding management options and environmental concerns. The agricultural valorization of unconventional organic materials has become inevitable, especially in semi-arid and arid countries that suffer from depleted soils and shortages in farm manure supply. High-income countries have also been interested in this recycling practice to mitigate landfilling or incineration issues. Sewage and some industrial sludges contain a complex mixture of beneficial and harmful substances, which varies with the origin of effluents. Therefore, sludge land application should be well managed in order to achieve sustainable agro-environmental goals. This review paper focuses on different aspects related to sludge reuse in agriculture, starting by investigating the diversity of sludge types and composition. In addition to the preponderant urban sewage sludge, the less-studied industrial sludges, such as those generated from pulp and paper mills or gas-to-liquid industries, are hereby addressed as well. Then, post-land application effects are discussed in relation to sludge quality, dose, and reuse conditions. The present paper also examines the disparities between guidelines that determine sludge conformity for land application in various countries or regions. Accordingly, special attention is given to increasing risks related to emerging pollutants in sludge such as pharmaceuticals, which have been overused since the outbreak of COVID-19 pandemic. This exhaustive investigation will assist the establishment of sustainable strategies for the safe agricultural reuse of biosolids.

The Influence of Municipal Wastewater Treatment Technologies on the Biological Stabilization of Sewage Sludge: A Systematic Review

Various wastewater treatment technologies are available today and biological processes are predominantly used in these technologies. Increasing wastewater treatment systems produces large amounts of sewage sludge with variable quantities and qualities, which must be properly managed. Anaerobic and aerobic digestion and composting are major strategies to treat this sludge. The main indicators of biological stabilization are volatile fatty acids (VFAs), volatile solids (VS), the carbon/nitrogen (C/N) ratio, humic substances (HS), the total organic carbon (TOC), the carbon dioxide (CO2) evolution rate, the specific oxygen uptake rate (SOUR), and the Dewar test;however, different criteria exist for the same indicators. Although there is no consensus for defining the stability of sewage sludge (biosolids) in the research and regulations reviewed, controlling the biological degradation, vector attraction, and odor determines the biological stabilization of sewage sludge. Because pollutants and pathogens are not completely removed in biological stabilization processes, further treatments to improve the quality of biosolids and to ensure their safe use should be explored.

Occurrence of viruses in sewage sludge: A systematic review.

Enteric viruses are of great importance in wastewater due to their high excretion from infected individuals, low removal in wastewater treatment processes, long-time survival in the environment, and low infectious dose. Among the other viruses, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) surveillance in wastewater systems has received particular attention as a result of the current COVID-19 epidemic. Viruses adhering to solid particles in wastewater treatment processes will end up as sewage sludge, and therefore insufficient sludge treatment may result in viral particles dissemination into the environment. Here, we review data on viruses' presence in sewage sludge, their detection and concentration methods, and information on human health issues associated with sewage sludge land application. We used combinations of the following keywords in the Scopus, Web of Science (WOS), and PubMed databases, which were published between 2010 and January 21th, 2022: sludge (sewage sludge, biosolids, sewage solids, wastewater solids) and virus (enteric virus, viral particles, viral contamination, SARS-CoV-2, coronavirus). The sources were searched twice, once with and then without the common enteric virus names (adenovirus, rotavirus, norovirus, enterovirus, hepatitis A virus). Studies suggest adenovirus and norovirus as the most prevalent enteric viruses in sewage sludge. Indeed, other viruses include rotavirus, hepatitis A virus, and enterovirus were frequently found in sewage sludge samples. Untreated biological sludge and thickened sludge showed more viral contamination level than digested sludge and the lowest prevalence of viruses was reported in lime stabilized sludge. The review reveals that land application of sewage sludge may pose viral infection risks to people due to accidently ingestion of sludge or intake of crops grown in biosolids amended soil. Moreover, contamination of groundwater and/or surface water may occur due to land application of sewage sludge.

Comprehensive investigation of SARS-CoV-2 fate in wastewater and finding the virus transfer and destruction route through conventional activated sludge and sequencing batch reactor.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA transmission route was thoroughly investigated in the hospital wastewater, sewage collection network, and wastewater treatment plants. Samples were taken on four occasions from December 2020 to April 2021. The performance of two different wastewater treatment processes of sequencing batch reactor (SBR) and conventional activated sludge (CAS) was studied for virus destruction. For this purpose, liquid phase, solid phase and bioaerosol samples were taken from different units of the investigated wastewater treatment plants (WWTPs). The results revealed that all untreated hospital wastewater samples were positive for SARS-CoV-2 RNA. The virus detection frequency increased when the number of hospitalized cases increased. Detection of viral RNA in the wastewater collection system exhibited higher load of virus in the generated wastewater in areas with poor socioeconomic conditions. Virus detection in the emitted bioaerosols in WWTPs showed that bioaerosols released from CAS with surface aeration contains SARS-CoV-2 RNA posing a potential threat to the working staff of the WWTPs. However, no viral RNA was detected in the bioaerosols of the SBR with diffused aeration system. Investigation of SARS-CoV-2 RNA in WWTPs showed high affinity of the virus to be accumulated in biosolids rather than transporting via liquid phase. Following the fate of virus in sludge revealed that it is completely destructed in anaerobic sludge treatment process. Therefore, based on the results of the present study, it can be concluded that receiving water resources could not be contaminated with virus, if the wastewater treatment processes work properly.

Prevalence of SARS-CoV-2 genes in water reclamation facilities: From influent to anaerobic digester.

Several treatment plants were sampled for influent, primary clarifier sludge, return activated sludge (RAS), and anaerobically digested sludge throughout nine weeks during the summer of the COVID-19 pandemic. Primary clarifier sludge had a significantly higher number of SARS-CoV-2 gene copy number per liter (GC/L) than other sludge samples, within a range from 1.0 × 105 to 1.0 × 106 GC/L. Gene copy numbers in raw influent significantly correlated with gene copy numbers in RAS in Silver Creek (p-value = 0.007, R2 = 0.681) and East Canyon (p-value = 0.009, R2 = 0.775) WRFs; both of which lack primary clarifiers or industrial pretreatment processes. This data indicates that SARS-CoV-2 gene copies tend to partition into primary clarifier sludges, at which point a significant portion of them are removed through sedimentation. Furthermore, it was found that East Canyon WRF gene copy numbers in influent were a significant predictor of daily cases (p-value = 0.0322, R2 = 0.561), and gene copy numbers in RAS were a significant predictor of weekly cases (p-value = 0.0597, R2 = 0.449). However, gene copy numbers found in primary sludge samples from other plants significantly predicted the number of COVID-19 cases for the following week (t = 2.279) and the week after that (t = 2.122) respectively. These data indicate that SARS-CoV-2 extracted from WRF biosolids may better suit epidemiological monitoring that exhibits a time lag. It also supports the observation that primary sludge removes a significant portion of SARS-CoV-2 marker genes. In its absence, RAS can also be used to predict the number of COVID-19 cases due to direct flow through from influent. This research represents the first of its kind to thoroughly examine SARS-CoV-2 gene copy numbers in biosolids throughout the wastewater treatment process and the relationship between primary, return activated, and anaerobically digested sludge and reported positive COVID-19 cases.

Assessing and managing SARS-CoV-2 occupational health risk to workers handling residuals and biosolids.

Current wastewater worker guidance from the United States Environmental Protection Agency (USEPA) aligns with the Centers for Disease Control and Prevention (CDC) and the Occupational Safety and Health Administration (OSHA) recommendations and states that no additional specific protections against SARS-CoV-2, the virus that causes COVID-19 infections, are recommended for employees involved in wastewater management operations with residuals, sludge, and biosolids at water resource recovery facilities. The USEPA guidance references a document from 2002 that summarizes practices required for protection of workers handling class B biosolids to minimize exposure to pathogens including viruses. While there is no documented evidence that residuals or biosolids of any treatment level contain infectious SARS-CoV-2 or are a source of transmission of this current pandemic strain of coronavirus, this review summarizes and examines whether the provided federal guidance is sufficient to protect workers in view of currently available data on SARS-CoV-2 persistence and transmission. No currently available epidemiological data establishes a direct link between wastewater sludge or biosolids and risk of infection from the SARS-CoV-2. Despite shedding of the RNA of the virus in feces, there is no evidence supporting the presence or transmission of infectious SARS-CoV-2 through the wastewater system or in biosolids. In addition, this review presents previous epidemiologic data related to other non-enveloped viruses. Overall, the risk for exposure to SARS-CoV-2, or any pathogen, decreases with increasing treatment measures. As a result, the highest risk of exposure is related to spreading and handling untreated feces or stool, followed by untreated municipal sludge, the class B biosolids, while lowest risk is associated with spreading or handling Class A biosolids. This review reinforces federal recommendations and the importance of vigilance in applying occupational risk mitigation measures to protect public and occupational health.