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1. Long-read metagenomic sequencing reveals shifts in associations of antibiotic resistance genes with mobile genetic elements from sewage to activated sludge

   https://doi.org/10.1186/s40168-021-01216-5  

   Dai, Dongjuan; Brown, Connor; Bürgmann, Helmut; Larsson, D. G. Joakim; Nambi, Indumathi; Zhang, Tong; Flach, Carl-Fredrik; Pruden, Amy; Vikesland, Peter J. (January 2022, Microbiome)

   Abstract BackgroundThere is concern that the microbially rich activated sludge environment of wastewater treatment plants (WWTPs) may contribute to the dissemination of antibiotic resistance genes (ARGs). We applied long-read (nanopore) sequencing to profile ARGs and their neighboring genes to illuminate their fate in the activated sludge treatment by comparing their abundance, genetic locations, mobility potential, and bacterial hosts within activated sludge relative to those in influent sewage across five WWTPs from three continents. ResultsThe abundances (gene copies per Gb of reads, aka gc/Gb) of all ARGs and those carried by putative pathogens decreased 75–90% from influent sewage (192-605 gc/Gb) to activated sludge (31-62 gc/Gb) at all five WWTPs. Long reads enabled quantification of the percent abundance of ARGs with mobility potential (i.e., located on plasmids or co-located with other mobile genetic elements (MGEs)). The abundance of plasmid-associated ARGs decreased at four of five WWTPs (from 40–73 to 31–68%), and ARGs co-located with transposable, integrative, and conjugative element hallmark genes showed similar trends. Most ARG-associated elements decreased 0.35–13.52% while integrative and transposable elements displayed slight increases at two WWTPs (1.4–2.4%). While resistome and taxonomic compositions both shifted significantly, host phyla for chromosomal ARG classes remained relatively consistent, indicating vertical gene transfer via active biomass growth in activated sludge as the key pathway of chromosomal ARG dissemination. ConclusionsOverall, our results suggest that the activated sludge process acted as a barrier against the proliferation of most ARGs, while those that persisted or increased warrant further attention. 

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2. Comparative assessment of pre‐ and inter‐stage hydrothermal treatment of municipal sludge for increased methane production

   https://doi.org/10.1002/wer.1523  

   Zhang, Chiqian; Liu, Xiaoguang; Wang, Qian; Tang, Yuanzhi; Pavlostathis, Spyros G. (March 2021, Water Environment Research)

   Abstract Hydrothermal treatment (HT) is a promising technology to enhance anaerobic digestion (AD) of municipal sludge. However, the capacity of pre‐ and inter‐stage HT (i.e., HT‐AD and AD‐HT‐AD, respectively) to enhance the digestibility of municipal sludge has not been sufficiently explored. This study compared the efficacy of pre‐ and inter‐stage HT performed from 90 to 185°C to enhance methane production from a mixture of primary sludge and waste activated sludge using mesophilic (35°C) biochemical methane potential tests. In both configurations, sludge solubilization increased with HT temperature. HT‐AD, and to a greater extent AD‐HT‐AD, increased the release of ammonium nitrogen. Even though HT at 185°C dramatically increased sludge solubilization, the overall specific methane yield with HT at 185°C was lower than or comparable to that at lower HT temperatures in the HT‐AD and AD‐HT‐AD configurations, respectively. Up to 155°C HT, the overall specific methane yield with the HT‐AD configuration was higher by 4.9%–8.3% compared to the AD‐HT‐AD configuration. However, when the HT energy was considered, compared to the control (i.e., AD of sludge without HT), the net energy gain (ΔE) decreased as the HT temperature increased, becoming negative at an HT of 185°C. The AD‐HT‐AD configuration resulted in a higher overall volatile solids destruction (by 8.1 to 20.1%). In conclusion, for municipal sludge with a relatively high ultimate digestibility, as was the case in this study, HT‐AD is preferable as it has a smaller footprint and is easier to operate than the AD‐HT‐AD configuration. However, given the significantly higher volatile solids destruction in the AD‐HT‐AD configuration, compared to the HT‐AD configuration, AD‐HT‐AD may be more beneficial considering post‐AD sludge handling processes. Practitioner pointsHydrothermal treatment (HT) increased the rate and extent of methane production from municipal sludge mixture.155°C was the optimal temperature for either pre‐ or inter‐stage HT to increase biogas production.Pre‐ and inter‐stage HT resulted in comparable ultimate methane production.Pre‐stage HT is preferable to inter‐stage HT (smaller footprint, easier to operate).AD‐HT‐AD resulted in significantly higher volatile solids destruction compared to the HT‐AD configuration. 

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3. Meta-omic profiling reveals ubiquity of genes encoding for the nitrogen-rich biopolymer cyanophycin in activated sludge microbiomes

   https://doi.org/10.3389/fmicb.2023.1287491  

   Farmer, McKenna; Rajasabhai, Rashmi; Tarpeh, William; Tyo, Keith; Wells, George (November 2023, Frontiers in Microbiology)

   Recovering nitrogen (N) from municipal wastewater is a promising approach to prevent nutrient pollution, reduce energy use, and transition toward a circular N bioeconomy, but remains a technologically challenging endeavor. Existing N recovery techniques are optimized for high-strength, low-volume wastewater. Therefore, developing methods to concentrate dilute N from mainstream wastewater will bridge the gap between existing technologies and practical implementation. The N-rich biopolymer cyanophycin is a promising candidate for N bioconcentration due to its pH-tunable solubility characteristics and potential for high levels of accumulation. However, the cyanophycin synthesis pathway is poorly explored in engineered microbiomes. In this study, we analyzed over 3,700 publicly available metagenome assembled genomes (MAGs) and found that the cyanophycin synthesis genecphAwas ubiquitous across common activated sludge bacteria. We found thatcphAwas present in common phosphorus accumulating organisms (PAO)Ca.‘Accumulibacter’ andTetrasphaera,suggesting potential for simultaneous N and P bioconcentration in the same organisms. Using metatranscriptomic data, we confirmed the expression ofcphAin lab-scale bioreactors enriched with PAO. Our findings suggest that cyanophycin synthesis is a ubiquitous metabolic activity in activated sludge microbiomes. The possibility of combined N and P bioconcentration could lower barriers to entry for N recovery, since P concentration by PAO is already a widespread biotechnology in municipal wastewater treatment. We anticipate this work to be a starting point for future evaluations of combined N and P bioaccumulation, with the ultimate goal of advancing widespread adoption of N recovery from municipal wastewater. 

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

   https://doi.org/doi.org/10.1016/j.scitotenv.2021.148905  

   Bishav Bhattarai; Sierra Quinn Sahulka; Aditi Podder; Soklida Hong; Hanyan Li; Eddie Gilcrease; Alex Beams; Rebecca Steed; and Ramesh Goel* (July 2021, Science of the total environment)

   Not Known (Ed.)

   Several treatment plants were sampled for raw 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 1.0x105 to 1.0x106 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). 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. 

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5. Atrazine Removal from Municipal Wastewater Using a Membrane Bioreactor

   https://doi.org/10.3390/ijerph17072567  

   Kamaz, Mohanad; Jones, Steven M.; Qian, Xianghong; Watts, Michael J.; Zhang, Wen; Wickramasinghe, S. Ranil (April 2020, International Journal of Environmental Research and Public Health)

   As the demand for potable water increases, direct potable reuse of wastewater is an attractive alternative method to produce potable water. However, implementation of such a process will require the removal of emerging contaminants which could accumulate in the drinking water supply. Here, the removal of atrazine, a commonly used herbicide, has been investigated. Using real and synthetic wastewater, as well as sludge from two wastewater treatment facilities in the United States in Norman, Oklahoma and Fayetteville, Arkansas, atrazine removal has been investigated. Our results indicate that about 20% of the atrazine is removed by adsorption onto the particulate matter present. Significant biodegradation of atrazine was only observed under aerobic conditions for sludge from Norman, Oklahoma. Next-generation sequencing of the activated sludge revealed the abundance of Noncardiac with known atrazine degradation pathways in the Norman aerobic sludge, which is believed to be responsible for atrazine biodegradation in our study. The detection of these bacteria could also be used to determine the likelihood of biodegradation of atrazine for a given wastewater treatment facility. 

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