Balancing public health and group privacy: Ethics, rights, and obligations for wastewater surveillance systems.

As the threat of COVID-19 recedes, wastewater surveillance - unlike other pandemic-era public health surveillance methods - seems here to stay. Concerns have been raised, however, about the potential risks that wastewater surveillance might pose towards group privacy. Existing scholarship has focused upon using ethics- or human rights-based frameworks as a means of balancing the public health objectives of wastewater surveillance and the potential risks it might pose to group privacy. However, such frameworks greatly lack enforceability. In order to further the strong foundation laid by such frameworks - while addressing their lack of enforceability - this paper proposes the idea of the 'obligation' as an alternative way to regulate wastewater surveillance systems. The legal codification of said obligations provides a method of ensuring that wastewater surveillance systems can be deployed effectively and equitably. Our paper proposes that legal obligations for wastewater surveillance can be created and enforced through transparent and purposeful legislation (which would include limits on power and grant institutions substantial oversight) as well as paying heed to non-legislative legal means of enforcement, such as through courts or contracts. Introducing legal obligations for wastewater surveillance could therefore be highly useful to researchers, policymakers, corporate technologists, and government agencies working in this field.

Microbial community-based protein from soybean-processing wastewater as a sustainable alternative fish feed ingredient.

As the global demand for food increases, aquaculture plays a key role as the fastest growing animal protein sector. However, existing aquafeeds contain protein ingredients that are not sustainable under current production systems. We evaluated the use of microbial community-based single cell protein (SCP), produced from soybean processing wastewater, as a partial fishmeal protein substitute in juvenile Asian seabass (Lates calcarifer). A 24-day feeding trial was conducted with a control fishmeal diet and a 50% fishmeal replacement with microbial community-based SCP as an experimental group, in triplicate tanks containing 20 fish each. Both diets met the protein, essential amino acids (except for lysine), and fat requirements for juvenile Asian sea bass. The microbial composition of the SCP was dominated by the genera Acidipropionibacterium and Propioniciclava, which have potential as probiotics and producers of valuable metabolites. The growth performance in terms of percent weight gain, feed conversion ratio (FCR), specific growth rate (SGR), and survival were not significantly different between groups after 24 days. The experimental group had less variability in terms of weight gain and FCR than the control group. Overall, microbial community-based protein produced from soybean processing wastewater has potential as a value-added feed ingredient for sustainable aquaculture feeds.

Molecular detection and viability discrimination of zoonotic protozoan pathogens in oysters and seawater.

The presence of foodborne protozoan pathogens including Cryptosporidium parvum, Giardia duodenalis, Toxoplasma gondii, and Cyclospora cayetanensis in commercial shellfish has been reported across diverse geographical regions. In the present study, a novel multiplex nested polymerase chain reaction (PCR) assay was validated to simultaneously detect and discriminate these four targeted parasites in oyster tissues including whole tissue homogenate, digestive gland, gills, and hemolymph, as well as seawater where shellfish grow. To differentiate viable and non-viable protozoan (oo)cysts, we further evaluated reverse transcription quantitative PCR (RT-qPCR) assays through systematic laboratory spiking experiments by spiking not only dilutions of viable parasites but also mixtures of viable and non-viable parasites in the oyster tissues and seawater. Results demonstrate that multiplex PCR can detect as few as 5-10 (oo)cysts in at least one oyster matrix, as well as in 10 L of seawater. All parasites were detected at the lowest spiking dilution (5 (oo)cysts per extract) in hemolymph, however the probability of detection varied across the difference matrices tested for each parasite. RT-qPCR further discriminated viable from non-viable (heat-inactivated) C. parvum and T. gondii in seawater and hemolymph but did not perform well in other oyster matrices. This systematic spiking study demonstrates that a molecular approach combining multiplex PCR for sensitive and affordable screening of protozoan DNA and subsequent RT-qPCR assay for viability discrimination presents an important advance for accurately determining the risk of protozoal illness in humans due to consumption of contaminated shellfish.

Metagenomics and metatranscriptomics suggest pathways of 3-chloroaniline degradation in wastewater reactors.

Biological wastewater treatment systems are often affected by shifts in influent quality, including the input of toxic chemicals. Yet the mechanisms underlying the adaptation of activated sludge process performance are rarely studied in a controlled and replicated experimental setting, particularly when challenged with a sustained toxin input. Three replicate bench-scale bioreactors were subjected to a chemical disturbance in the form of 3-chloroaniline (3-CA) over 132 days, after an acclimation period of 58 days, while three control reactors received no 3-CA input. Ammonia oxidation was initially affected by 3-CA. Within three weeks of the experiment, microbial communities in all three treatment reactors adapted to biologically degrade 3-CA resulting in partial ammonia oxidation recovery. Combining process and microbial community data from amplicon sequencing with potential functions gleaned from assembled metagenomics and metatranscriptomics data, two putative degradation pathways for 3-CA were identified. The first pathway, determined from metagenomics data, involves a benzoate dioxygenase and subsequent meta-cleavage of the aromatic ring. The second, determined from intensive short-term sampling for gene expression data in tandem with 3-CA degradation, involves a phenol monooxygenase followed by ortho-cleavage of the aromatic ring. The relative abundances of amplicon sequence variants associated with the genera Gemmatimonas, OLB8, and Taibaiella correlated significantly with 3-CA degradation. Metagenome-assembled genome data also showed the genus OLB8 to be differentially enriched in treatment reactors, making it a strong candidate as 3-CA degrader. Using replicated reactors, this study has demonstrated the impact of a sustained stress on the activated sludge process. The unique and novel features of this study include the identification of putative pathways and potential degraders of 3-CA using long-term and short-term sampling in tandem with multiple methods in a controlled and replicated experiment.

CRISPR-Cas phage defense systems and prophages in Candidatus Accumulibacter.

Candidatus Accumulibacter plays a major role in enhanced biological phosphorus removal (EBPR) from wastewater. Although bacteriophages have been shown to represent fatal threats to Ca. Accumulibacter organisms and thus interfere with the stability of the EBPR process, little is known about the ability of different Ca. Accumulibacter strains to resist phage infections. We conducted a systematic analysis of the occurrence and characteristics of clustered regularly interspaced short palindromic repeats and associated proteins (CRISPR-Cas) systems and prophages in Ca. Accumulibacter lineage members (43 in total, including 10 newly recovered genomes). Results indicate that 28 Ca. Accumulibacter genomes encode CRISPR-Cas systems. They were likely acquired via horizontal gene transfer, conveying a distinct adaptivity to phage predation to different Ca. Accumulibacter members. Major differences in the number of spacers show the unique phage resistance of these members. A comparison of the spacers in closely related Ca. Accumulibacter members from distinct geographical locations indicates that habitat isolation may have resulted in the acquisition of resistance to different phages by different Ca. Accumulibacter. Long-term operation of three laboratory-scale EBPR bioreactors revealed high relative abundances of Ca. Accumulibacter with CRISPSR-Cas systems. Their specific resistance to phages in these reactors was indicated by spacer analysis. Metatranscriptomic analyses showed the activation of the CRISPR-Cas system under both anaerobic and aerobic conditions. Additionally, 133 prophage regions were identified in 43 Ca. Accumulibacter genomes. Twenty-seven of them (in 19 genomes) were potentially active. Major differences in the occurrence of CRISPR-Cas systems and prophages in Ca. Accumulibacter will lead to distinct responses to phage predation. This study represents the first systematic analysis of CRISPR-Cas systems and prophages in the Ca. Accumulibacter lineage, providing new perspectives on the potential impacts of phages on Ca. Accumulibacter and EBPR systems.

Co-incidence of BA.1 and BA.2 at the start of Singapore's Omicron wave revealed by Community and University Campus wastewater surveillance.

Wastewater surveillance (WWS) has been globally recognised to be a useful tool in quantifying SARS-CoV-2 RNA at the community and residential levels without biases associated with case-reporting. The emergence of variants of concern (VOCs) have given rise to an unprecedented number of infections even though populations are increasingly vaccinated. This is because VOCs have been reported to possess higher transmissibility and can evade host immune responses. The B.1.1.529 lineage (Omicron) has severely disrupted global plans to return to normalcy. In this study, we developed an allele-specific (AS) RT-qPCR assay which simultaneously targets the stretch of deletions and mutations in the spike protein from position 24-27 for quantitative detection of Omicron BA.2. Together with previous assays that detect mutations associated with Omicron BA.1 (deletion at position 69 and 70) and all Omicron (mutation at position 493 and 498), we report the validation and time series of these assays from September 2021 to May 2022 using influent samples from two wastewater treatment plants and across four University campus sites in Singapore. Viral RNA concentrations at the treatment plants corroborate with locally reported clinical cases, AS RT-qPCR assays revealed co-incidence of Omicron BA.1 and BA.2 on 12 January 2022, almost two months after initial BA.1 detection in South Africa and Botswana. BA.2 became the dominant variant by the end of January 2022 and completely displaced BA.1 by mid-March 2022. University campus sites were similarly positive for BA.1 and/or BA.2 in the same week as first detection at the treatment plants, where BA.2 became rapidly established as the dominant lineage within three weeks. These results corroborate clinical incidence of the Omicron lineages in Singapore and indicate minimal silent circulation prior to January 2022. The subsequent simultaneous spread of both variant lineages followed strategic relaxation of safe management measures upon meeting nationwide vaccination goals.

Microbial community-based production of single cell protein from soybean-processing wastewater of variable chemical composition.

The use of food-processing wastewaters to produce microbial biomass-derived single cell protein (SCP) is a sustainable way to meet the global food demand. Microbial community-based approaches to SCP production have the potential benefits of lower costs and greater resource recovery compared to pure cultures, yet they have received scarce attention. Here, SCP production from soybean-processing wastewaters using their existent microbial communities was evaluated. Six sequencing batch reactors of 4.5-L working volume were operated at 30 °C for 34 d in cycles consisting of 3-h anaerobic and 9-h aerobic phases. Four reactors received no microbial inoculum and the remaining two were amended with 1.5 L of a mixed culture from a prior SCP production cycle. Reactors produced more SCP when fed with wastewaters of higher soluble total Kjeldahl nitrogen (sTKN) content. The protein yield in biomass ranged from 0.53 to 3.13 g protein/g sTKN, with a maximum protein content of 50 %. The average removal of soluble chemical oxygen demand (sCOD) and soluble total nitrogen (sTN) was 92 % and 73 %, respectively. Distinct microbial genera were enriched in all six bioreactors, with Azospirillum, Rhodobacter, Lactococcus, and Novosphingobium dominating. The study showed that constituents in soybean wastewater can be converted to SCP and demonstrated the effect of variable influent wastewater composition on SCP production.

Contextualizing Wastewater-Based surveillance in the COVID-19 vaccination era.

SARS-CoV-2 wastewater-based surveillance (WBS) offers a tool for cost-effective oversight of a population's infections. In the past two years, WBS has proven to be crucial for managing the pandemic across different geographical regions. However, the changing context of the pandemic due to high levels of COVID-19 vaccination warrants a closer examination of its implication towards SARS-CoV-2 WBS. Two main questions were raised: 1) Does vaccination cause shedding of viral signatures without infection? 2) Does vaccination affect the relationship between wastewater and clinical data? To answer, we review historical reports of shedding from viral vaccines in use prior to the COVID-19 pandemic including for polio, rotavirus, influenza and measles infection and provide a perspective on the implications of different COVID-19 vaccination strategies with regard to the potential shedding of viral signatures into the sewershed. Additionally, we reviewed studies that looked into the relationship between wastewater and clinical data and how vaccination campaigns could have affected the relationship. Finally, analyzing wastewater and clinical data from the Netherlands, we observed changes in the relationship concomitant with increasing vaccination coverage and switches in dominant variants of concern. First, that no vaccine-derived shedding is expected from the current commercial pipeline of COVID-19 vaccines that may confound interpretation of WBS data. Secondly, that breakthrough infections from vaccinated individuals contribute significantly to wastewater signals and must be interpreted in light of the changing dynamics of shedding from new variants of concern.

How can we possibly resolve the planet's nitrogen dilemma?

Nitrogen is the most crucial element in the production of nutritious feeds and foods. The production of reactive nitrogen by means of fossil fuel has thus far been able to guarantee the protein supply for the world population. Yet, the production and massive use of fertilizer nitrogen constitute a major threat in terms of environmental health and sustainability. It is crucial to promote consumer acceptance and awareness towards proteins produced by highly effective microorganisms, and their potential to replace proteins obtained with poor nitrogen efficiencies from plants and animals. The fact that reactive fertilizer nitrogen, produced by the Haber Bosch process, consumes a significant amount of fossil fuel worldwide is of concern. Moreover, recently, the prices of fossil fuels have increased the cost of reactive nitrogen by a factor of 3 to 5 times, while international policies are fostering the transition towards a more sustainable agro-ecology by reducing mineral fertilizers inputs and increasing organic farming. The combination of these pressures and challenges opens opportunities to use the reactive nitrogen nutrient more carefully. Time has come to effectively recover used nitrogen from secondary resources and to upgrade it to a legal status of fertilizer. Organic nitrogen is a slow-release fertilizer, it has a factor of 2.5 or higher economic value per unit nitrogen as fertilizer and thus adequate technologies to produce it, for instance by implementing photobiological processes, are promising. Finally, it appears wise to start the integration in our overall feed and food supply chains of the exceptional potential of biological nitrogen fixation. Nitrogen produced by the nitrogenase enzyme, either in the soil or in novel biotechnology reactor systems, deserves to have a 'renaissance' in the context of planetary governance in general and the increasing number of people who desire to be fed in a sustainable way in particular.

The biofilm matrix: multitasking in a shared space.

The biofilm matrix can be considered to be a shared space for the encased microbial cells, comprising a wide variety of extracellular polymeric substances (EPS), such as polysaccharides, proteins, amyloids, lipids and extracellular DNA (eDNA), as well as membrane vesicles and humic-like microbially derived refractory substances. EPS are dynamic in space and time and their components interact in complex ways, fulfilling various functions: to stabilize the matrix, acquire nutrients, retain and protect eDNA or exoenzymes, or offer sorption sites for ions and hydrophobic substances. The retention of exoenzymes effectively renders the biofilm matrix an external digestion system influencing the global turnover of biopolymers, considering the ubiquitous relevance of biofilms. Physico-chemical and biological interactions and environmental conditions enable biofilm systems to morph into films, microcolonies and macrocolonies, films, ridges, ripples, columns, pellicles, bubbles, mushrooms and suspended aggregates - in response to the very diverse conditions confronting a particular biofilm community. Assembly and dynamics of the matrix are mostly coordinated by secondary messengers, signalling molecules or small RNAs, in both medically relevant and environmental biofilms. Fully deciphering how bacteria provide structure to the matrix, and thus facilitate and benefit from extracellular reactions, remains the challenge for future biofilm research.

Controlling anammox speciation and biofilm attachment strategy using N-biotransformation intermediates and organic carbon levels.

Conventional nitrogen removal in wastewater treatment requires a high oxygen and energy input. Anaerobic ammonium oxidation (anammox), the single-step conversion of ammonium and nitrite to nitrogen gas, is a more energy and cost effective alternative applied extensively to sidestream wastewater treatment. It would also be a mainstream treatment option if species diversity and physiology were better understood. Anammox bacteria were enriched up to 80%, 90% and 50% relative abundance, from a single inoculum, under standard enrichment conditions with either stepwise-nitrite and ammonia concentration increases (R1), nitric oxide supplementation (R2), or complex organic carbon from mainstream wastewater (R3), respectively. Candidatus Brocadia caroliniensis predominated in all reactors, but a shift towards Ca. Brocadia sinica occurred at ammonium and nitrite concentrations > 270 mg NH4-N L-1 and 340 mg NO2-N L-1 respectively. With NO present, heterotrophic growth was inhibited, and Ca. Jettenia coexisted with Ca. B. caroliniensis before diminishing as nitrite increased to 160 mg NO2-N L-1. Organic carbon supplementation led to the emergence of heterotrophic communities that coevolved with Ca. B. caroliniensis. Ca. B. caroliniensis and Ca. Jettenia preferentially formed biofilms on surfaces, whereas Ca. Brocadia sinica formed granules in suspension. Our results indicate that multiple anammox bacteria species co-exist and occupy sub-niches in anammox reactors, and that the dominant population can be reversibly shifted by, for example, changing nitrogen load (i.e. high nitrite concentration favors Ca. Brocadia caroliniensis). Speciation has implications for wastewater process design, where the optimum cell immobilization strategy (i.e. carriers vs granules) depends on which species dominates.

The one health perspective to improve environmental surveillance of zoonotic viruses: lessons from COVID-19 and outlook beyond.

The human population has doubled in the last 50 years from about 3.7 billion to approximately 7.8 billion. With this rapid expansion, more people live in close contact with wildlife, livestock, and pets, which in turn creates increasing opportunities for zoonotic diseases to pass between animals and people. At present an estimated 75% of all emerging virus-associated infectious diseases possess a zoonotic origin, and outbreaks of Zika, Ebola and COVID-19 in the past decade showed their huge disruptive potential on the global economy. Here, we describe how One Health inspired environmental surveillance campaigns have emerged as the preferred tools to monitor human-adjacent environments for known and yet to be discovered infectious diseases, and how they can complement classical clinical diagnostics. We highlight the importance of environmental factors concerning interactions between animals, pathogens and/or humans that drive the emergence of zoonoses, and the methodologies currently proposed to monitor them-the surveillance of wastewater, for example, was identified as one of the main tools to assess the spread of SARS-CoV-2 by public health professionals and policy makers during the COVID-19 pandemic. One-Health driven approaches that facilitate surveillance, thus harbour the potential of preparing humanity for future pandemics caused by aetiological agents with environmental reservoirs. Via the example of COVID-19 and other viral diseases, we propose that wastewater surveillance is a useful complement to clinical diagnosis as it is centralized, robust, cost-effective, and relatively easy to implement.

Selective Enrichment of Nitrososphaera viennensis-Like Ammonia-Oxidizing Archaea over Ammonia-Oxidizing Bacteria from Drinking Water Biofilms.

Ammonia-oxidizing archaea (AOA) can oxidize ammonia to nitrite for energy gain. They have been detected in chloraminated drinking water distribution systems (DWDS) along with the more common ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB). To date, no members of the AOA have been isolated or enriched from drinking water environments. To begin the investigation of the role of AOA in chloraminated DWDS, we developed a selective approach using biofilm samples from a full-scale operational network as inoculum. A Nitrososphaera viennensis-like AOA taxon was enriched from a mixed community that also included Nitrosomonas-like AOB while gradually scaling up the culture volume. Dimethylthiourea (DMTU) and pyruvate at 100 µM were added to promote the growth of AOA while inhibiting AOB. This resulted in the eventual washout of AOB, while NOB were absent after 2 or 3 rounds of amendment with 24 µM sodium azide. The relative abundance of AOA in the enrichment increased from 0.2% to 39.5% after adding DMTU and pyruvate, and further to 51.6% after filtration through a 0.45-µm pore size membrane, within a period of approximately 6 months. IMPORTANCE Chloramination has been known to increase the risk of nitrification episodes in DWDS due to the presence of ammonia-oxidizing microorganisms. Among them, AOB are more frequently detected than AOA. All publicly available cultures of AOA have been isolated from soil, marine or surface water environments, meaning they are allochthonous to DWDS. Hence, monochloramine exposure studies involving these strains may not accurately reflect their role in DWDS. The described method allows for the rapid enrichment of autochthonous AOA from drinking water nitrifying communities. The high relative abundance of AOA in the resulting enrichment culture reduces any confounding effects of co-existing heterotrophic bacteria when investigating the response of AOA to varied levels of monochloramine in drinking water.

Monitoring human arboviral diseases through wastewater surveillance: Challenges, progress and future opportunities.

Arboviral diseases are caused by a group of viruses spread by the bite of infected arthropods. Amongst these, dengue, Zika, west nile fever and yellow fever cause the greatest economic and social impact. Arboviral epidemics have increased in frequency, magnitude and geographical extent over the past decades and are expected to continue increasing with climate change and expanding urbanisation. Arboviral prevalence is largely underestimated, as most infections are asymptomatic, nevertheless existing surveillance systems are based on passive reporting of loosely defined clinical syndromes with infrequent laboratory confirmation. Wastewater-based surveillance (WBS), which has been demonstrated to be useful for monitoring diseases with significant asymptomatic populations including COVID19 and polio, could be a useful complement to arboviral surveillance. We review the current state of knowledge and identify key factors that affect the feasibility of monitoring arboviral diseases by WBS to include viral shedding loads by infected persons, the persistence of shed arboviruses and the efficiency of their recovery from sewage. We provide a simple model on the volume of wastewater that needs to be processed for detection of arboviruses, in face of lower arboviral shedding rates. In all, this review serves to reflect on the key challenges that need to be addressed and overcome for successful implementation of arboviral WBS.

Resource recovery from food-processing wastewaters in a circular economy: a methodology for the future.

Food-processing wastewater (FPWW), as opposed to solid residues, occurs extensively throughout the world, and has considerable potential for resource recovery (RR), however, at present, it is severely underutilized. This paper serves as a suggested 'plan forward' to optimize integrated RR from FPWW. In order to optimize this potential and 'close the loop', there needs to be further development in in-depth analytical methods of the FPWW; axenic/mixed cultures or microbial communities capable of growing on FPWW and hence producing single-cell protein for animal feed and food additives; cost-effective methods for separating high-value-added solutes such as vitamins K2, B12, and B2; isoflavones and flavanones; integrated energy- and water-recovery flowsheets; and optimization methods to integrate RR and energy harvesting with minimal impact on the environment. Each of these areas is examined and future research directions are laid out.

Recovery of High Quality Metagenome-Assembled Genomes From Full-Scale Activated Sludge Microbial Communities in a Tropical Climate Using Longitudinal Metagenome Sampling.

The analysis of metagenome data based on the recovery of draft genomes (so called metagenome-assembled genomes, or MAG) has assumed an increasingly central role in microbiome research in recent years. Microbial communities underpinning the operation of wastewater treatment plants are particularly challenging targets for MAG analysis due to their high ecological complexity, and remain important, albeit understudied, microbial communities that play ssa key role in mediating interactions between human and natural ecosystems. Here we consider strategies for recovery of MAG sequence from time series metagenome surveys of full-scale activated sludge microbial communities. We generate MAG catalogs from this set of data using several different strategies, including the use of multiple individual sample assemblies, two variations on multi-sample co-assembly and a recently published MAG recovery workflow using deep learning. We obtain a total of just under 9,100 draft genomes, which collapse to around 3,100 non-redundant genomic clusters. We examine the strengths and weaknesses of these approaches in relation to MAG yield and quality, showing that co-assembly may offer advantages over single-sample assembly in the case of metagenome data obtained from closely sampled longitudinal study designs. Around 1,000 MAGs were candidates for being considered high quality, based on single-copy marker gene occurrence statistics, however only 58 MAG formally meet the MIMAG criteria for being high quality draft genomes. These findings carry broader broader implications for performing genome-resolved metagenomics on highly complex communities, the design and implementation of genome recoverability strategies, MAG decontamination and the search for better binning methodology.

Microbiome assembly predictably shapes diversity across a range of disturbance frequencies in experimental microcosms.

Diversity is often implied to have a positive effect on the functional stability of ecological communities. However, its relationship with stochastic and deterministic assembly mechanisms remains largely unknown, particularly under fluctuating disturbances. Here, we subjected complex bacterial communities in microcosms to different frequencies of alteration in substrate feeding scheme, tracking temporal dynamics in their assembly, structure and function. Activated sludge bioreactors were subjected to six different frequencies of double organic loading, either never (undisturbed), every 8, 6, 4, or 2 days (intermediately disturbed), or every day (press disturbed), and operated in daily cycles for 42 days. Null modeling revealed a stronger role of stochastic assembly at intermediate disturbance frequencies, with a peak in stochasticity that preceded the occurrence of a peak in α-diversity. Communities at extreme ends of the disturbance range had the lowest α-diversity and highest within-treatment similarity in terms of ß-diversity, with stronger deterministic assembly. Increased carbon removal and microbial aggregate settleability (general functions) correlated with stronger deterministic processes. In contrast, higher stochasticity correlated with higher nitrogen removal (a specialized function) only during initial successional stages at intermediate disturbance frequencies. We show that changes in assembly processes predictably precede changes in diversity under a gradient of disturbance frequencies, advancing our understanding of the mechanisms behind disturbance-diversity-function relationships.

Making waves: Wastewater surveillance of SARS-CoV-2 in an endemic future.

Wastewater-based surveillance (WBS) has been widely used as a public health tool to monitor the emergence and spread of SARS-CoV-2 infections in populations during the COVID-19 pandemic. Coincident with the global vaccination efforts, the world is also enduring new waves of SARS-CoV-2 variants. Reinfections and vaccine breakthroughs suggest an endemic future where SARS-CoV-2 continues to persist in the general population. In this treatise, we aim to explore the future roles of wastewater surveillance. Practically, WBS serves as a relatively affordable and non-invasive tool for mass surveillance of SARS-CoV-2 infection while minimizing privacy concerns, attributes that make it extremely suited for its long-term usage. In an endemic future, the utility of WBS will include 1) monitoring the trend of viral loads of targets in wastewater for quantitative estimate of changes in disease incidence; 2) sampling upstream for pinpointing infections in neighborhoods and at the building level; 3) integrating wastewater and clinical surveillance for cost-efficient population surveillance; and 4) genome sequencing wastewater samples to track circulating and emerging variants in the population. We further discuss the challenges and future developments of WBS to reduce inconsistencies in wastewater data worldwide, improve its epidemiological inference, and advance viral tracking and discovery as a preparation for the next viral pandemic.

Global warming readiness: Feasibility of enhanced biological phosphorus removal at 35 °C.

Recent research has shown enhanced biological phosphorus removal (EBPR) from municipal wastewater at warmer temperatures around 30 °C to be achievable in both laboratory-scale reactors and full-scale treatment plants. In the context of a changing climate, the feasibility of EBPR at even higher temperatures is of interest. We operated two lab-scale EBPR sequencing batch reactors for > 300 days at 30 °C and 35 °C, respectively, and followed the dynamics of the communities of polyphosphate accumulating organisms (PAOs) and competing glycogen accumulating organisms (GAOs) using a combination of 16S rRNA gene metabarcoding, quantitative PCR and fluorescence in situ hybridization analyses. Stable and nearly complete phosphorus (P) removal was achieved at 30 °C; similarly, long term P removal was stable at 35 °C with effluent PO43-_P concentrations < 0.5 mg/L on half of all monitored days. Diverse and abundant Candidatus Accumulibacter amplicon sequence variants were closely related to those found in temperate environments, suggesting that EBPR at this temperature does not require a highly specialized PAO community. A slow-feeding strategy effectively limited the carbon uptake rates of GAOs, allowing PAOs to outcompete GAOs at both temperatures. Candidatus Competibacter was the main GAO, along with cluster III Defluviicoccus members. These organisms withstood the slow-feeding regime, suggesting that their bioenergetic characteristics of carbon uptake differ from those of their tetrad-forming relatives. Comparative cycle studies revealed higher carbon and P cycling activity of Ca. Accumulibacter when the temperature was increased from 30 °C to 35 °C, implying that the lowered P removal performance at 35 °C was not a direct effect of temperature, but a result of higher metabolic rates of carbon (and/or P) utilization of PAOs and GAOs, the resultant carbon deficiency, and escalated community competition. An increase in the TOC-to-PO43--P ratio (from 25:1 to 40:1) effectively eased the carbon deficiency and benefited PAOs. In general, a slow-feeding strategy and sufficiently high carbon input benefited a high and stable EBPR at 35 °C, representing basic conditions suitable for full-scale treatment plants experiencing higher water temperatures.

Development of an efficient wastewater testing protocol for high-throughput country-wide SARS-CoV-2 monitoring.

Wastewater-based surveillance has been widely used as a non-intrusive tool to monitor population-level transmission of COVID-19. Although various approaches are available to concentrate viruses from wastewater samples, scalable methods remain limited. Here, we sought to identify and evaluate SARS-CoV-2 virus concentration protocols for high-throughput wastewater testing. A total of twelve protocols for polyethylene glycol (PEG) precipitation and four protocols for ultrafiltration-based approaches were evaluated across two phases. The first phase entailed an initial evaluation using a small sample set, while the second phase further evaluated five protocols using wastewater samples of varying SARS-CoV-2 concentrations. Permutations in the pre-concentration, virus concentration and RNA extraction steps were evaluated. Among PEG-based methods, SARS-CoV-2 virus recovery was optimal with 1) the removal of debris prior to processing, 2) 2 h to 24 h incubation with 8% PEG at 4 °C, 3) 4000 xg or 14,000 xg centrifugation, and 4) a column-based RNA extraction method, yielding virus recovery of 42.4-52.5%. Similarly, the optimal protocol for ultrafiltration included 1) the removal of debris prior to processing, 2) ultrafiltration, and 3) a column-based RNA extraction method, yielding a recovery of 38.2%. This study also revealed that SARS-CoV-2 RNA recovery for samples with higher virus concentration were less sensitive to changes in the PEG method, but permutations in the PEG protocol could significantly impact virus yields when wastewater samples with lower SARS-CoV-2 RNA were used. Although both PEG precipitation and ultrafiltration methods resulted in similar SARS-CoV-2 RNA recoveries, the former method is more cost-effective while the latter method provided operational efficiency as it required a shorter turn-around-time (PEG precipitation, 9-23 h; Ultrafiltration, 5 h). The decision on which method to adopt will thus depend on the use-case for wastewater testing, and the need for cost-effectiveness, sensitivity, operational feasibility and scalability.