Evaluation of low-cost SARS-CoV-2 RNA purification methods for viral quantification by RT-qPCR and next-generation sequencing analysis: Implications for wider wastewater-based epidemiology adoption.

Based Epidemiology (WBE) consists of quantifying biomarkers in sewerage systems to derive real-time information on the health and/or lifestyle of the contributing population. WBE usefulness was vastly demonstrated in the context of the COVID-19 pandemic. Many methods for SARS-CoV-2 RNA determination in wastewater were devised, which vary in cost, infrastructure requirements and sensitivity. For most developing countries, implementing WBE for viral outbreaks, such as that of SARS-CoV-2, proved challenging due to budget, reagent availability and infrastructure constraints. In this study, we assessed low-cost methods for SARS-CoV-2 RNA quantification by RT-qPCR, and performed variant identification by NGS in wastewater samples. Results showed that the effect of adjusting pH to 4 and/or adding MgCl2 (25 mM) was negligible when using the adsorption-elution method, as well as basal physicochemical parameters in the sample. In addition, results supported the standardized use of linear rather than plasmid DNA for a more accurate viral RT-qPCR estimation. The modified TRIzol-based purification method in this study yielded comparable RT-qPCR estimation to a column-based approach, but provided better NGS results, suggesting that column-based purification for viral analysis should be revised. Overall, this work provides evaluation of a robust, sensitive and cost-effective method for SARS-CoV-2 RNA analysis that could be implemented for other viruses, for a wider WEB adoption.

Spatiotemporal Surveillance of SARS-CoV-2 in the Sewage of Three Major Urban Areas in Peru: Generating Valuable Data Where Clinical Testing Is Extremely Limited.

Peru has been severely affected by the COVID-19 pandemic. By January 2022, Peru had surpassed 200 000 COVID-19 deaths, constituting the highest death rate per capita worldwide. Peru has had several limitations during the pandemic: insufficient testing access, limited contact tracing, a strained medical infrastructure, and many economic hurdles. These limitations hindered the gathering of accurate information about infected individuals with spatial resolution in real time, a critical aspect of effectively controlling the pandemic. Wastewater monitoring for SARS-CoV-2 RNA offered a promising alternative for providing needed population-wide information to complement health care indicators. In this study, we demonstrate the feasibility and value of implementing a decentralized SARS-CoV-2 RNA wastewater monitoring system to assess the spatiotemporal distribution of COVID-19 in three major cities in Peru: Lima, Callao, and Arequipa. Our data on viral loads showed the same trends as health indicators such as incidence and mortality. Furthermore, we were able to identify hot spots of contagion within the surveyed urban areas to guide the efforts of health authorities. Viral decay in the sewage network of the cities studied was found to be negligible (<2%). Overall, our results support wastewater monitoring for SARS-CoV-2 as a valuable and cost-effective tool for monitoring the COVID-19 pandemic in the Peruvian context.

Assessing spatial distribution of COVID-19 prevalence in Brazil using decentralised sewage monitoring.

Brazil has become one of the epicentres of the COVID-19 pandemic, with cases heavily concentrated in large cities. Testing data is extremely limited and unreliable, which restricts health authorities' ability to deal with the pandemic. Given the stark demographic, social and economic heterogeneities within Brazilian cities, it is important to identify hotspots so that the limited resources available can have the greatest impact. This study shows that decentralised monitoring of SARS-CoV-2 RNA in sewage can be used to assess the distribution of COVID-19 prevalence in the city. The methodology developed in this study allowed the identification of hotspots by comprehensively monitoring sewers distributed through Belo Horizonte, Brazil's third largest city. Our results show that the most vulnerable neighbourhoods in the city were the hardest hit by the pandemic, indicating that, for many Brazilians, the situation is much worse than reported by official figures.

Occurrence and removal of micropollutants in full-scale aerobic, anaerobic and facultative wastewater treatment plants in Brazil.

This study aims to evaluate micropollutant occurrence and removal in a low-middle income country (LMIC) by investigating the occurrence of 28 chemicals from different classes (triclosan, 15 polycyclic aromatic hydrocarbons (PAHs), 4 estrogens and 8 polybrominated diphenyl ether (PBDE) congeners) in three technologically diverse full-scale Brazilian wastewater treatment plants (WWTPs). These chemicals were detected at concentrations similar to those reported in other low-middle income countries (LMICs) and high-income countries (HICs) (0.1-49 µg/L) indicating their widespread use globally and the need for more studies in LMICs that are typically characterized by relatively inadequate wastewater treatment barriers. Among the three different WWTPs investigated for removal of these chemicals, the least energy intensive system, waste stabilization ponds (WSPs), was the most effective (95-99%) compared to the activated sludge (79-94%), and Up-flow sludge blanket reactor (UASB) with trickling filters system (89-95%). These results highlight the potential of WSPs for micropollutant removal-especially in warm climates. However, the effluent from all three WWTP could pose a risk to aquatic organisms when discharged into the receiving waters as the effluent concentrations of triclosan, some estrogens, PAHs and BDE 209 were above European environmental quality standards (EQS) or predicted no effect concentration (PNEC values), indicating that receiving water bodies could benefit from further treatment. In combination, these results help to further understand prevailing concentrations of micropollutants globally and fate in current wastewater treatment systems.

Assessment of the performance of an anoxic-aerobic microalgal-bacterial system treating digestate.

The performance of an anoxic-aerobic microalgal-bacterial system treating synthetic food waste digestate at 10 days of hydraulic retention time via nitrification-denitrification under increasing digestate concentrations of 25%, 50%, and 100% (v/v) was assessed during Stages I, II and III, respectively. The system supported adequate treatment without external CO2 supplementation since sufficient inorganic carbon in the digestate was available for autotrophic growth. High steady-state Total Organic Carbon (TOC) and Total Nitrogen (TN) removal efficiencies of 85-96% and 73-84% were achieved in Stages I and II. Similarly, PO43--P removals of 81 ± 15% and 58 ± 4% were recorded during these stages. During Stage III, the average influent concentrations of 815 ± 35 mg TOC·L-1, 610 ± 23 mg TN·L-1, and 46 ± 11 mg PO43--P·L-1 induced O2 limiting conditions, resulting in TOC, TN and PO43--P removals of 85 ± 3%, 73 ± 3%, and 28 ± 16%, respectively. Digestate concentrations of 25% and 50% favored nitrification-denitrification mechanisms, whereas the treatment of undiluted digestate resulted in higher ammonia volatilization and hampered nitrification-denitrification. In Stages I and II, the microalgal community was dominated by Chlorella vulgaris and Cryptomonas sp., whereas Pseudoanabaena sp. was more abundant during Stage III. Illumina sequencing revealed the presence of carbon and nitrogen transforming bacteria, with dominances of the genera Gemmata, Azospirillum, and Psychrobacter during Stage I, II, and III, respectively. Finally, the high settleability of the biomass (98% of suspended solids removal in the settler) and average C (42%), N (7%), P (0.2%), and S (0.4%) contents recovered in the biomass confirmed its potential for agricultural applications, contributing to a closed-cycle management of food waste.

Background data on solar heat-assisted stripping-absorption system for ammonia recovery from food waste digestate.

The data presented in this paper are related to the research article "Ammonia recovery from food waste digestate using solar heat-assisted stripping-absorption" [1]. The raw and filtered data are associated to daily monitoring of NH4 concentration of food waste digestate, pH of digestate and absorption solution and temperature of food waste digestate throughout experiments at different conditions. In addition, data of temperature monitoring in different points of solar-heat assisted stripping-absorption device are presented. The data could help further studies aiming to improve this system. The detailed data of these experiments could help to improve the performance and to reduce costs of nitrogen recovery from digestate using stripping-absorption technology.

Ammonia recovery from food waste digestate using solar heat-assisted stripping-absorption.

The highest costs of stripping-absorption processes for ammonia recovery are related to energy (for heating and air supply) and chemical addition (for pH adjustment). In this paper, a simplified system that used no chemicals, and a renewable source of energy for heating, was tested to recover nitrogen as ammonium sulfate from food waste digestate. pH adjustment was achieved by CO2 stripping, and vacuum tube solar collectors were used to provide heating. The effect of different temperatures (25 °C and 45 °C) and gas to liquid ratios (1700 and 2600) on ammonia removal and recovery were assessed. Ammonia removal efficiencies higher than 91% were achieved for all evaluated experimental conditions. The solar heater showed adequate capacity to increase the temperature of the liquid digestate by 21 °C and maintain the temperature at 45 °C throughout the experiment. Tests carried out at 45 °C achieved the highest ammonia removal efficiency (98%) at the lowest evaluated G/L ratio (1700). Better absorption efficiencies could potentially have been achieved if lower inlet airflow rates and packing material had been used in the absorption column.

COVID-19 faecal-oral transmission: Are we asking the right questions?

Detection of the SARS-CoV-2 virus in stools and sewage has recently been reported, raising the hypothesis of faecal-oral transmission. If confirmed, this could have far-reaching consequences for public health and for pandemic control strategies. In this paper, we argue that a comprehensive and more nuanced analysis is required to test this hypothesis, taking into consideration both environmental dynamics and the persistence of viral infectivity. First, we examine the evidence regarding the presence of the virus in stools and sewage. Then we discuss the current framework of disease transmission through water and excreta and how the transmission of a respiratory disease fits into it. Against this background, we propose a framework to test the faecal-oral hypothesis, unpacking the different environmental routes from faeces to the mouth of a susceptible person. This framework should not be seen as a confirmation of the hypothesis but rather as an expanded view of its complexities, which could help shaping an agenda for research into a number of unanswered questions. Finally, the paper briefly discusses practical implications, based on current knowledge, for containment of the pandemic.

Polyhydroxyalkanoates (PHA) production from biogas in waste treatment facilities: Assessing the potential impacts on economy, environment and society.

Using the biogas generated from organic waste anaerobic treatment to produce polyhydroxyalkanoates (PHAs) has emerged as an attractive alternative to heat and power generation (CHP) in waste treatment plants. The sustainability of biogas combustion for CHP, biogas bioconversion into PHA and a combination of both scenarios was compared in terms of environmental impact, process economics and social responsibility according to the IChemE Sustainability Metrics. Although PHA production presented higher investment and operational costs, a comparable economic performance was observed in all biogas valorization scenarios regarding net present value (0.77 M€) and internal rate of return (6.4 ± 0.2%) due to the higher market value of biopolymers. The PHA production entailed a significant reduction of atmospheric acidification and odor emissions compared to CHP despite showing higher land, water, chemicals and energy requirements. Job creation associated to biopolymer industry and the increasing public demand for bioproducts were identified as fundamental aspects for enhancing social and local acceptance of waste processing facilities. This study demonstrated that PHA production from biogas constitutes nowadays a realistic alternative to CHP in waste treatment plants and that PHA can be produced at a competitive market price when biogas is used for internal energy provision (4.2 €·kg-1 PHA).

Upflow anaerobic sludge blanket in microalgae-based sewage treatment: Co-digestion for improving biogas production.

Upflow anaerobic sludge blanket (UASB) reactors are widely used to treat domestic sewage and frequently require post-treatment. Little is known about the use of high rate algal ponds (HRAP) for post-treating UASB reactors' effluent. This study aimed to evaluate a UASB reactor followed by a HRAP in terms of sewage treatment efficiency and biogas production, during one year at demonstration-scale. The UASB reactor co-treated raw sewage and the harvested microalgal biomass from the HRAP, which was recirculated to the reactor. An identical UASB reactor, treating only raw sewage, was used as control. The results showed an overall removal of 65% COD and 61% N-NH4 in the system. Furthermore, methane yield was increased by 25% after anaerobic co-digestion with microalgae, from 156 to 211 NL CH4 kg-1 VS. An energy assessment was performed and showed a positive energy balance, with a net ratio of 2.11 to the annual average.

Structural integrity affects nitrogen removal activity of granules in semi-continuous reactors.

Granules were observed after more than two years of operation in two semi-continuously fed intermittently aerated reactors treating swine wastewater with aerobic:anoxic cycles of 1:1 h and 1:4 h. Subsequently, the granules and flocs were compared with respect to physical characteristics, activity, and microbial community structure. Granules exhibited higher specific nitrification and denitrification rates than flocs. However, once granule structural integrity was disrupted, the rates decreased to levels similar to those of flocs. Membrane hybridizations using 16S rRNA-targeted probes showed that ammonia oxidizing bacteria populations in flocs and granules were dominated by Nitrosomonas and Nitrosococcus mobilis. Granules provided better conditions for Nitrospira compared to flocs. The diversities of the dominant bacterial populations in granules and flocs were not significantly different. Our findings highlight the importance of structural integrity of granules to their nitrogen removing activity.

Evaluation of DNA extraction methods for freshwater eukaryotic microalgae.

The use of molecular methods to investigate microalgal communities of natural and engineered freshwater resources is in its infancy, with the majority of previous studies carried out by microscopy. Inefficient or differential DNA extraction of microalgal community members can lead to bias in downstream community analysis. Three commercially available DNA extraction kits have been tested on a range of pure culture freshwater algal species with diverse cell walls and mixed algal cultures taken from eutrophic waste stabilization ponds (WSP). DNA yield and quality were evaluated, along with DNA suitability for amplification of 18S rRNA gene fragments by polymerase chain reaction (PCR). QiagenDNeasy(®) Blood and Tissue kit (QBT), was found to give the highest DNA yields and quality. Denaturant Gradient Gel Electrophoresis (DGGE) was used to assess the diversity of communities from which DNA was extracted. No significant differences were found among kits when assessing diversity. QBT is recommended for use with WSP samples, a conclusion confirmed by further testing on communities from two tropical WSP systems. The fixation of microalgal samples with ethanol prior to DNA extraction was found to reduce yields as well as diversity and is not recommended.

Identification of nitrite-reducing bacteria using sequential mRNA fluorescence in situ hybridization and fluorescence-assisted cell sorting.

Sequential mRNA fluorescence in situ hybridization (mRNA FISH) and fluorescence-assisted cell sorting (SmRFF) was used for the identification of nitrite-reducing bacteria in mixed microbial communities. An oligonucleotide probe labeled with horseradish peroxidase (HRP) was used to target mRNA of nirS, the gene that encodes nitrite reductase, the enzyme responsible for the dissimilatory reduction of nitrite to nitric oxide. Clones for nirS expression were constructed and used to provide proof of concept for the SmRFF method. In addition, cells from pure cultures of Pseudomonas stutzeri and denitrifying activated sludge were hybridized with the HRP probe, and tyramide signal amplification was performed, conferring a strongly fluorescent signal to cells containing nirS mRNA. Flow cytometry-assisted cell sorting was used to detect and physically separate two subgroups from a mixed microbial community: non-fluorescent cells and an enrichment of fluorescent, nitrite-reducing cells. Denaturing gradient gel electrophoresis (DGGE) and subsequent sequencing of 16S ribosomal RNA (rRNA) genes were used to compare the fragments amplified from the two sorted subgroups. Sequences from bands isolated from DGGE profiles suggested that the dominant, active nitrite reducers were closely related to Acidovorax BSB421. Furthermore, following mRNA FISH detection of nitrite-reducing bacteria, 16S rRNA FISH was used to detect ammonia-oxidizing and nitrite-oxidizing bacteria on the same activated sludge sample. We believe that the molecular approach described can be useful as a tool to help address the longstanding challenge of linking function to identity in natural and engineered habitats.