Natural influent bioaugmentation of activated sludge water resource recovery systems: implications for low temperature nitrification and heterotrophic population structures

This work aimed at demonstrating the natural bioaugmentation of biological activated sludge systems with nitrifying biomass from influent wastewater in lab-scale reactors. Three sequencing batch reactors (SBR) were fed with sterile synthetic wastewater. While nitrification was complete at a temperature of 8 °C and a SRT of 20 days, it failed when the temperature was lowered to 5 °C, and the SRT decreased to 7 days. In the test period, the sterile synthetic wastewater fed to the Test Reactor was supplemented by influent solids harvested at a full-scale treatment facility at a total suspended solids concentration of 100 mg/L, which corresponded to approximately 5 mg-COD/L of nitrifying biomass. Upon this addition, nitrification was restores. Subsequent halting the supply of influent solids to the Test Reactor led a rapid failure of nitrification and washout of nitrifiers from the SBR. Reproducibility was demonstrated by switching the feed composition between the Test and Negative control reactors. PCR-based amplicon sequencing analyses targeting the amoA, and nxrB genes of the Nitrospira genus have shown that the influent wastewater governed the structure and composition of the activated sludge nitrifying populations. The most abundant ammonia-oxidizing bacteria (AOB) and Nitrospira-related nitrite-oxidizing bacteria (NOB) in the influent seeds occurred as the most dominant ones in the activated sludge. This pattern was observed even when the influent seeds varied over time. The heterotrophic populations were less affected by the influent seeds with the activated sludge and raw sewage showing distinct microbial populations based on principal coordinate analysis (PCoA). However, the immigrant populations appeared to modulate the structure of the activated sludge heterotrophic communities to some extent. These findings provide concrete evidence of the presence of active nitrifiers in raw wastewater capable of supporting nitrification in an otherwise non-conducive environment. This may have important implications on process design, operation and optimization of wastewater treatment systems.HighlightsLab-scale reactors fed sterile synthetic wastewater at low temperatures and SRTs.Nitrification failed when conditions were adjusted to 5 °C and a SRT of 7 days.Nitrification restored by addition of real wastewater influent solids.Nitrifiers in solids from sewers naturally bioaugment activated sludge systems.Activated sludge models should consider the immigration of nitrifiers with influent.Graphical abstract

Tracking the temporal variation of COVID-19 surges through wastewater-based epidemiology during the peak of the pandemic: a six-month long study in Charlotte, North Carolina

The global spread of SARS-CoV-2 has continued to be a serious concern after WHO declared the virus the causative agent of the coronavirus disease 2019 (COVID-19) a global pandemic. Monitoring of wastewater is a useful tool for assessing community prevalence given that fecal shedding of SARS-CoV-2 occurs in high concentrations by infected individuals, regardless of whether they are asymptomatic or symptomatic. Using tools that are part of the wastewater-based epidemiology (WBE) approach, combined with molecular analyses, wastewater monitoring becomes a key piece of information used to assess trends and quantify the scale and dynamics of COVID-19 infection in a specific community, municipality, or area of service. This study investigates a six-month long SARS-CoV-2 RNA quantification in influent wastewater from four municipal wastewater treatment plants (WWTP) serving the Charlotte region of North Carolina (NC) using both RT-qPCR and RT-ddPCR platforms. Influent wastewater was analyzed for the nucleocapsid (N) genes N1 and N2. Both RT-qPCR and RT-ddPCR performed well for detection and quantification of SARS-CoV-2 using the N1 target, while for the N2 target RT-ddPCR was more sensitive. SARS-CoV-2 concentration ranged from 103 to105 copies/L for all four plants. Both RT-qPCR and RT-ddPCR showed a significant moderate to a strong positive correlation between SARS-CoV-2 concentrations and the 7-day rolling average of clinically reported COVID-19 cases using a lag that ranged from 7 to 12 days. A major finding of this study is that despite small differences, both RT-qPCR and RT-ddPCR performed well for tracking the SARS-CoV-2 virus across WWTP of a range of sizes and metropolitan service functions.

Assessing multiplex tiling PCR sequencing approaches for detecting genomic variants of SARS-CoV-2 in municipal wastewater

Wastewater-based genomic surveillance of the SARS-CoV-2 virus shows promise to complement genomic epidemiology efforts. Multiplex tiled PCR is a desirable approach for targeted genome sequencing of SARS-CoV-2 in wastewater due to its low cost and rapid turnaround time. However, it is not clear how different multiplex tiled PCR primer schemes or wastewater sample matrices impact the resulting SARS-CoV-2 genome coverage. The objective of this work was to assess the performance of three different multiplex primer schemes, consisting of 150bp, 400bp, and 1200bp amplicons, as well as two wastewater sample matrices, influent wastewater and primary sludge, for targeted genome sequencing of SARS-CoV-2. Wastewater samples were collected weekly from five municipal wastewater treatment plants (WWTPs) in the Metro Vancouver region of British Columbia, Canada during a period of increased COVID-19 case counts from February to April, 2021. RNA extracted from clarified influent wastewater provided significantly higher genome coverage (breadth and median depth) than primary sludge samples across all primer schemes. Shorter amplicons appeared more resilient to sample RNA degradation, but were hindered by greater primer pool complexity in the 150bp scheme. The identified optimal primer scheme (400bp) and sample matrix (influent) was capable of detecting the emergence of mutations associated with genomic variants of concern, of which the daily wastewater load significantly correlated with clinical case counts. Taken together, these results provide guidance on best practices for implementing wastewater-based genomic surveillance, and demonstrate its ability to inform epidemiology efforts by detecting genomic variants of concern circulating within a geographic region.

Urban wastewater bacterial communities assemble into seasonal steady states

Background Microorganisms in urban sanitary sewers exhibit community properties that suggest sewers are a novel ecosystem. Sewer microorganisms present both an opportunity as a control point for wastewater treatment and a risk to human health. If treatment processes are to be improved and health risks quantified, then it is necessary to understand microbial distributions and dynamics within this community. Here, we use 16S rRNA gene sequencing to characterize raw influent wastewater bacterial communities in a 5-year time series from two wastewater treatment plants in Milwaukee, WI; influent wastewater from 77 treatment plants across the USA; and wastewater in 12 Milwaukee residential sewers. Results In Milwaukee, we find that in transit from residences to treatment plants, the human bacterial component of wastewater decreases in proportion and exhibits stochastic temporal variation. In contrast, the resident sewer community increases in abundance during transit and cycles seasonally according to changes in wastewater temperature. The result is a bacterial community that assembles into two distinct community states each year according to the extremes in wastewater temperature. Wastewater bacterial communities from other northern US cities follow temporal trends that mirror those in Milwaukee, but southern US cities have distinct community compositions and differ in their seasonal patterns. Conclusions Our findings provide evidence that environmental conditions associated with seasonal change and climatic differences related to geography predictably structure the bacterial communities residing in below-ground sewer pipes.

Assessing multiplex tiling PCR sequencing approaches for detecting genomic variants of SARS-CoV-2 in municipal wastewater v1

In this work, we aim to access the performance of three different multiplex primer schemes, i.e. Swift amplicon SARS-CoV-2 panel (150bp amplicons), ARTIC V3 panel (400bp amplicons), and SARS-CoV-2 midnight panel (1200bp amplicons), for metatranscriptomic sequencing of SARS-CoV-2 for influent wastewater and primary sludge. This protocol is adapted from the Swift amplicon™ SARS-COV-2 protocol (150bp amplicon), ARTIC V3 protocol (400bp amplicon), and "midnight" protocol (1200bp amplicon). Sequencing libraries are prepared with 1) Oxford Nanopore Ligation Sequencing Kit (SQK-LSK109) with Native Barcoding kit (EXP-NEB104 and EXP-NEB114), or 2) NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® with NEBNext® Multiplex Oligos for Illumina®. Links to the protocols are: Swift amplicon protocol (150bp): https://swiftbiosci.com/swift-amplicon-sars-cov-2-panel/ ARTIC V3 protocol (400bp): https://www.protocols.io/view/ncov-2019-sequencing-protocol-v3-locost-bh42j8ye?version_warning=no "midnight" protocol V4 (1200bp): dx.doi.org/10.17504/protocols.io.bh7hj9j6

Reflections of COVID-19 cases on the wastewater loading of SARS-CoV-2 RNA: A case of three major cities of Gujarat, India

The scientific community has widely supported wastewater monitoring of SARS-CoV-2 due to the early and prolonged excretion of coronavirus in the faecal matter. In the present study, eighteen influent wastewater samples from different wastewater treatment plants and pumping stations (5 samples from Vadodara city, 4 from Gandhinagar, and nine from Ahmedabad city) were collected and analyzed for the occurrence of SARS-CoV-2 RNA in Gujarat province, India. The results showed the highest SARS-CoV-2 genome concentration in Vadodara (3078 copies/ L), followed by Ahmedabad (2968 copies/ L) and Gandhinagar (354 copies/ L). The comparison of genome concentration corresponded to the number of confirmed and active cases in all three cities. The study confirms the potential of the Surveillance of Wastewater for Early Epidemic Prediction (SWEEP) that can be used at a large scale around the globe for better dealing with the pandemic situation.

Is dairy wastewater safe for production of edible microbial biomass: A case study of Saras dairy plant at Jaipur, India

Mismanagement of wastewater at large scale may lead to catastrophic environmental and health consequences. Microbial remediation of wastewater is one of the most effective low-cost solutions. There are also initiatives to use wastewater for production edible biomass as an alternative for protein diets. While much researches were oriented towards maximum recovery of biomass and applications, less were focused on mutagenicity of dairy wastewater. In this study, we examined wastewater of one of the largest dairy industries in Rajasthan for its suitability for microbial biomass production and mutagenicity. Influent wastewater was collected from Saras dairy plant, Jaipur, for over a week. Physiochemical properties of wastewater were examined, such as; temperature, pH, salinity, TSS, TDS, turbidity, conductivity, BOD, COD, total carbon, and total nitrogen. SOS chromotest and Salmonella fluctuation test (TA 98, TA 100 and TA 102) were carried out at variable concentration of wastewater to assess mutagenic activity. Results indicated ideal pH, temperature and salinity, for microbial remediation. High TOC and TKN were also observed in the investigated wastewater, which is few of the prerequisites for single cell production. The ratio of BOD and COD was between 0.3-0.4, making the wastewater ideal for microbial growth. No mutagenic activity was observed by SOS chromotest, all three concentrations (C 0.01, C 0.1, and C 0.2) investigated in this study were <1.5 IF. Likewise, mutagenic ratio for all three types of Salmonella revertants were below 1.2 threshold, for investigated concentrations (C 0.5, C 1, and C 10) of wastewater. Conclusively, examined influent wastewater is less likely to induce mutagenic activity at the investigated concentration. Through physiochemical analysis, the investigated wastewater assumed to be candidate substrate for microbial biomass production.