Accounting for the microbial assembly of each process in wastewater treatment plants (WWTPs): study of four WWTPs receiving similar influent streams.

We evaluated a unique model in which four full-scale wastewater treatment plants (WWTPs) with the same treatment schematic and fed with similar influent wastewater were tracked over an 8-month period to determine whether the community assembly would differ in the activated sludge (AS) and sand filtration (SF) stages. For each WWTP, AS and SF achieved an average of 1-log10 (90%) and <0.02-log10 (5%) reduction of total cells, respectively. Despite the removal of cells, both AS and SF had a higher alpha and beta diversity compared to the influent microbial community. Using the Sloan neutral model, it was observed that AS and SF were individually dominated by different assembly processes. Specifically, microorganisms from influent to AS were predominantly determined by the selective niche process for all WWTPs, while the microbial community in the SF was relatively favored by a stochastic, random migration process, except two WWTPs. AS also contributed more to the final effluent microbial community compared with the SF. Given that each WWTP operates the AS independently and that there is a niche selection process driven mainly by the chemical oxygen demand concentration, operational taxonomic units unique to each of the WWTPs were also identified. The findings from this study indicate that each WWTP has its distinct microbial signature and could be used for source-tracking purposes.IMPORTANCEThis study provided a novel concept that microorganisms follow a niche assembly in the activated sludge (AS) tank and that the AS contributed more than the sand filtration process toward the final microbial signature that is unique to each treatment plant. This observation highlights the importance of understanding the microbial community selected by the AS stage, which could contribute toward source-tracking the effluent from different wastewater treatment plants.

Predicting Anaerobic Membrane Bioreactor Performance Using Flow-Cytometry-Derived High and Low Nucleic Acid Content Cells.

Having a tool to monitor the microbial abundances rapidly and to utilize the data to predict the reactor performance would facilitate the operation of an anaerobic membrane bioreactor (AnMBR). This study aims to achieve the aforementioned scenario by developing a linear regression model that incorporates a time-lagging mode. The model uses low nucleic acid (LNA) cell numbers and the ratio of high nucleic acid (HNA) to LNA cells as an input data set. First, the model was trained using data sets obtained from a 35 L pilot-scale AnMBR. The model was able to predict the chemical oxygen demand (COD) removal efficiency and methane production 3.5 days in advance. Subsequent validation of the model using flow cytometry (FCM)-derived data (at time t - 3.5 days) obtained from another biologically independent reactor did not exhibit any substantial difference between predicted and actual measurements of reactor performance at time t. Further cell sorting, 16S rRNA gene sequencing, and correlation analysis partly attributed this accurate prediction to HNA genera (e.g., Anaerovibrio and unclassified Bacteroidales) and LNA genera (e.g., Achromobacter, Ochrobactrum, and unclassified Anaerolineae). In summary, our findings suggest that HNA and LNA cell routine enumeration, along with the trained model, can derive a fast approach to predict the AnMBR performance.

UV and bacteriophages as a chemical-free approach for cleaning membranes from anaerobic bioreactors.

Anaerobic membrane bioreactor (AnMBR) for wastewater treatment has attracted much interest due to its efficacy in providing high-quality effluent with minimal energy costs. However, membrane biofouling represents the main bottleneck for AnMBR because it diminishes flux and necessitates frequent replacement of membranes. In this study, we assessed the feasibility of combining bacteriophages and UV-C irradiation to provide a chemical-free approach to remove biofoulants on the membrane. The combination of bacteriophage and UV-C resulted in better log cells removal and ca. 2× higher extracellular polymeric substance (EPS) concentration reduction in mature biofoulants compared to either UV-C or bacteriophage alone. The cleaning mechanism behind this combined approach is by 1) reducing the relative abundance of Acinetobacter spp. and selected bacteria (e.g., Paludibacter, Pseudomonas, Cloacibacterium, and gram-positive Firmicutes) associated with the membrane biofilm and 2) forming cavities in the biofilm to maintain water flux through the membrane. When the combined treatment was further compared with the common chemical cleaning procedure, a similar reduction on the cell numbers was observed (1.4 log). However, the combined treatment was less effective in removing EPS compared with chemical cleaning. These results suggest that the combination of UV-C and bacteriophage have an additive effect in biofouling reduction, representing a potential chemical-free method to remove reversible biofoulants on membrane fitted to an AnMBR.

Evaluation of protein extraction methods to improve meta-proteomics analysis of treated wastewater biofilms.

Metaproteomics can be used to study functionally active biofilm-based bacterial populations in reclaimed water distribution systems, which in turn result in bacterial regrowth that impacts the water quality. However, existing protein extraction methods have differences in their protein recovery and have not been evaluated for their efficacies in reclaimed water biofilm samples. In this study, we first evaluated six different protein extraction methods with diverse chemical and physical properties on a mixture of bacterial cell culture. Based on a weighting scores-based evaluation, the extraction protocols in order of decreasing performance are listed as B-PER > RIPA > PreOmics > SDS > AllPrep > Urea. The highest four optimal methods on cell culture were further tested against treated wastewater non-chlorinated and chlorinated effluent biofilms. In terms of protein yield, our findings showed that RIPA performed the best; however, the highest number of proteins were extracted from SDS and PreOmics. Furthermore, SDS and PreOmics worked best to rupture gram-positive and gram-negative bacterial cell walls. Considering the five evaluation factors, PreOmics obtained highest weighted score, indicating its potential effectiveness in extracting proteins from biofilms. This study provides the first insight into evaluating protein extraction methods to facilitate metaproteomics for complex reclaimed water matrices.

Growth dynamics and transcriptional responses of a Red Sea Prochlorococcus strain to varying temperatures.

Prochlorococcus play a crucial role in the ocean's biogeochemical cycling, but it remains controversial how they will respond to global warming. Here we assessed the response to temperature (22-30°C) of the growth dynamics and gene expression profiles of a Red Sea Prochlorococcus strain (RSP50) in a non-axenic culture. Both the specific growth rate (0.55-0.80 day-1 ) and cell size (0.04-0.07 µm3 ) of Prochlorococcus increased significantly with temperature. The primary production released extracellularly ranged from 20% to 34%, with humic-like fluorescent compounds increasing up to fivefold as Prochlorococcus reached its maximum abundance. At 30°C, genes involved in carbon fixation such as CsoS2 and CsoS3 and photosynthetic electron transport including PTOX were downregulated, suggesting a cellular homeostasis and energy saving mechanism response. In contrast, PTOX was found upregulated at 22°C and 24°C. Similar results were found for transaldolase, related to carbon metabolism, and citrate synthase, an important enzyme in the TCA cycle. Our data suggest that in spite of the currently warm temperatures of the Red Sea, Prochlorococcus can modulate its gene expression profiles to permit growth at temperatures lower than its optimum temperature (28°C) but is unable to cope with temperatures exceeding 30°C.

Novel Use of a Ferric Salt to Enhance Mainstream Nitrogen Removal from Anaerobically Pretreated Wastewater.

This study aims to demonstrate a new technology roadmap to support the ongoing paradigm shift in wastewater management from pollutant removal to resource recovery. This is achieved by developing a novel use of an iron salt (i.e., FeCl3) in an integrated anaerobic wastewater treatment and mainstream anammox process. FeCl3 was chosen to be dosed in a proposed sidestream unit rather than in a primary settler or a mainstream reactor. This causes acidification of returned activated sludge and enables stable suppression of nitrite-oxidizing bacterial activity and excess sludge reduction. A laboratory-scale system, which comprised an anaerobic baffled reactor, a continuous-flow anoxic-aerobic (A/O) reactor, and a secondary settler, was designed to treat real domestic wastewater, with the performance of the system comprehensively monitored under a steady-state condition. The experimental assessments showed that the system had good effluent quality, with total nitrogen and phosphorus concentrations of 12.6 ± 1.3 mg N/L and 0.34 ± 0.05 mg P/L, respectively. It efficiently retained phosphorus in excess sludge (0.18 ± 0.03 g P/g dry sludge), suggesting its potential for further recovery. About half of influent organic carbon was recovered in the form of bioenergy (i.e., methane). This together with low energy consumption revealed that the system could produce a net energy of about 0.11 kWh/m3-wastewater, assessed by an energy balance analysis.

Investigation of Antibiotic Resistome in Hospital Wastewater during the COVID-19 Pandemic: Is the Initial Phase of the Pandemic Contributing to Antimicrobial Resistance?

Since the COVID-19 pandemic started, there has been much speculation about how COVID-19 and antimicrobial resistance may be interconnected. In this study, untreated wastewater was sampled from Hospital A designated to treat COVID-19 patients during the first wave of the COVID-19 pandemic alongside Hospital B that did not receive any COVID-19 patients. Metagenomics was used to determine the relative abundance and mobile potential of antibiotic resistant genes (ARGs), prior to determining the correlation of ARGs with time/incidence of COVID-19. Our findings showed that ARGs resistant to macrolides, sulfonamides, and tetracyclines were positively correlated with time in Hospital A but not in Hospital B. Likewise, minor extended spectrum beta-lactamases (ESBLs) and carbapenemases of classes B and D were positively correlated with time, suggesting the selection of rare and/or carbapenem-resistant genes in Hospital A. Non-carbapenemase blaVEB also positively correlated with both time and intI1 and was copresent with other ARGs including carbapenem-resistant genes in 6 metagenome-assembled genomes (MAGs). This study highlighted concerns related to the dissemination of antimicrobial resistance (AMR) during the COVID-19 pandemic that may arise from antibiotic use and untreated hospital wastewater.

Recent Update on UV Disinfection to Fulfill the Disinfection Credit Value for Enteric Viruses in Water.

Ultraviolet (UV) radiation alone or in combination with other oxidation processes is increasingly being considered for water disinfection because of stringent regulatory requirements for pathogen inactivation. To fulfill this requirement, an appropriate UV dose or fluence (mJ/cm2) is applied to combat enteric viruses in surface or treated water. There is a need for a cumulative review on the effectiveness of current and emerging UV technologies against various types of human enteric viruses. We extracted the kinetics data from 52 selected experimental studies on enteric virus inactivation using low pressure (LP-UV), medium pressure (MP-UV), UV-LED, and advanced oxidation processes (AOPs) and applied a simple linear regression analysis to calculate the range of UV fluence (mJ/cm2) needed for 4-log10 inactivation. The inactivation of adenoviruses with LP-UV, MP-UV, and UV/H2O2 (10 mg/L) required the highest fluence, which ranged from 159 to 337, 45, and 115 mJ/cm2, respectively. By contrast, when using LP-UV, the inactivation of other enteric viruses, such as the Caliciviridae and Picornaviridae family and rotavirus, required fluence that ranged from 19 to 69, 18 to 43, and 38 mJ/cm2, respectively. ssRNA viruses exhibit higher sensitivity to UV radiation than dsRNA and DNA viruses. In general, as an upgrade to LP-UV, MP-UV is a more promising strategy for eliminating enteric viruses compared to AOP involving LP-UV with added H2O2 or TiO2. The UV-LED technology showed potential because a lower UV fluence (at 260 and/or 280 nm wavelength) was required for 4-log10 inactivation compared to that of LP-UV for most strains examined in this critical review. However, more studies evaluating the inactivation of enteric viruses by means of UV-LEDs and UV-AOP are needed to ascertain these observations.

Attached-growth configuration outperforms continuously stirred tank anaerobic membrane bioreactors in alleviating membrane biofouling.

Biofouling impedes the performance of anaerobic membrane bioreactors (AnMBR). Two reactors, one as an up-flow attachment-growth AnMBR (UA-AnMBR) configuration, and the other, as a continuously stirred AnMBR (CS-AnMBR) were evaluated for differences in membrane fouling rate. TMP increment in UA-AnMBR was slower than CS-AnMBR, although both reactors had similar COD removal efficiency (ca. > 96%). Slower fouling rate for UA-AnMBR was related to lower total and viable cells, and thereby microbial activity compared to that in CS-AnMBR. Acinetobacter and Methanobacterium that played keystone roles in anaerobic biofilm formation were not consistently prevalent on the membranes connected to UA-AnMBR. This is in contrast to both Acinetobacter and Methanobacterium consistently prevalent on the membranes connected to CS-AnMBR. The findings suggest that UA-AnMBR can alleviate membrane biofouling through changes in microbial activity and profile dynamics, and would be a suitable reactor configuration to adopt to achieve an efficient AnMBR for municipal wastewater treatment.

The use of UV/H2O2 to facilitate removal of emerging contaminants in anaerobic membrane bioreactor effluents.

Effluent from anaerobic membrane bioreactor (AnMBR) contains ammonia and would require post-polishing treatment before it can be disinfected by chlorine. However, additional post-treatment steps to remove nutrients offset the energetic benefits derived from anaerobic fermentation. The use of chlorine or ozone also promotes concerns associated with disinfection byproducts. This study evaluates UV/H2O2 as a potential strategy suited for the removal of pharmaceutical compounds as well as antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) from AnMBR effluent. Our findings indicate that 10 mg/L H2O2 and 61.5 mJ/cm2 of UV fluence are able to achieve a 4-log removal of both Escherichia coli PI7 and Klebsiella pneumoniae L7. However, a higher fluence of 311 mJ/cm2 with the same amount of H2O2 would be required to achieve >90% removal of atenolol, carbamazepine and estrone. The removal of the pharmaceutical compounds was driven by the hydroxyl radicals generated from H2O2, while UV exposure governed the inactivation of ARB and ARGs. UV/H2O2 increased overall mutagenicity of the treated wastewater matrix but did not result in any changes to the natural transformation rates. Instead, UV significantly reduced natural transformation rates by means of DNA damage. Overall, UV/H2O2 could be the ideal final disinfection strategy for AnMBR effluent without requiring additional post-treatment prior disinfection.

Estimating the minimum number of SARS-CoV-2 infected cases needed to detect viral RNA in wastewater: To what extent of the outbreak can surveillance of wastewater tell us?

There is increasing interest in wastewater-based epidemiology (WBE) of SARS-CoV-2 RNA to serve as an early warning system for a community. Despite successful detection of SARS-CoV-2 RNA in wastewaters sampled from multiple locations, there is still no clear idea on the minimal number of cases in a community that are associated with a positive detection of the virus in wastewater. To address this knowledge gap, we sampled wastewaters from a septic tank (n = 57) and biological activated sludge tank (n = 52) located on-site of a hospital. The hospital is providing treatment for SARS-CoV-2 infected patients, with the number of hospitalized patients per day known. It was observed that depending on which nucleocapsid gene is targeted by means of RT-qPCR, a range of 253-409 positive cases out of 10,000 persons are required prior to detecting RNA SARS-CoV-2 in wastewater. There was a weak correlation between N1 and N2 gene abundances in wastewater with the number of hospitalized cases. This correlation was however not observed for N3 gene. The frequency of detecting N1 and N2 gene in wastewater was also higher than that for N3 gene. Furthermore, nucleocapsid genes of SARS-CoV-2 were detected at lower frequency in the partially treated wastewater than in the septic tank. In particular, N1 gene abundance was associated with water quality parameters such as total organic carbon and pH. In instances of positive detection, the average abundance of N1 and N3 genes in the activated sludge tank were reduced by 50 and 70% of the levels detected in septic tank, suggesting degradation of the SARS-CoV-2 gene fragments already occurring in the early stages of the wastewater treatment process.

Metagenomics as a Tool To Monitor Reclaimed-Water Quality.

Many biological contaminants are disseminated through water, and their occurrence has potential detrimental impacts on public and environmental health. Conventional monitoring tools rely on cultivation and are not robust in addressing modern water quality concerns. This review proposes metagenomics as a means to provide a rapid, nontargeted assessment of biological contaminants in water. When further coupled with appropriate methods (e.g., quantitative PCR and flow cytometry) and bioinformatic tools, metagenomics can provide information concerning both the abundance and diversity of biological contaminants in reclaimed waters. Further correlation between the metagenomic-derived data of selected contaminants and the measurable parameters of water quality can also aid in devising strategies to alleviate undesirable water quality. Here, we review metagenomic approaches (i.e., both sequencing platforms and bioinformatic tools) and studies that demonstrated their use for reclaimed-water quality monitoring. We also provide recommendations on areas of improvement that will allow metagenomics to significantly impact how the water industry performs reclaimed-water quality monitoring in the future.

Metagenomics-based evaluation of groundwater microbial profiles in response to treated wastewater discharge.

This study aims to demonstrate the use of metagenomics to assess groundwater quality. Metagenomics revealed a lower alpha diversity for both bacteria and virus in wastewater-exposed groundwater compared to the upstream controls. An increase in the relative abundance of Planctomycetes and Picornaviridae was also observed in wastewater-exposed groundwater. However, comparison of antibiotic resistome cannot clearly differentiate wastewater-exposed groundwater from control. Findings suggest that metagenomics can detect selected microbial signatures indicative of treated wastewater discharge.

Effect of Quorum Sensing on the Ability of Desulfovibrio vulgaris To Form Biofilms and To Biocorrode Carbon Steel in Saline Conditions.

Sulfate-reducing bacteria (SRB) are key contributors to microbe-induced corrosion (MIC), which can lead to serious economic and environmental impact. The presence of a biofilm significantly increases the MIC rate. Inhibition of the quorum-sensing (QS) system is a promising alternative approach to prevent biofilm formation in various industrial settings, especially considering the significant ecological impact of conventional chemical-based mitigation strategies. In this study, the effect of the QS stimulation and inhibition on Desulfovibrio vulgaris is described in terms of anaerobic respiration, cell activity, biofilm formation, and biocorrosion of carbon steel. All these traits were repressed when bacteria were in contact with QS inhibitors but enhanced upon exposure to QS signal molecules compared to the control. The difference in the treatments was confirmed by transcriptomic analysis performed at different time points after treatment application. Genes related to lactate and pyruvate metabolism, sulfate reduction, electron transfer, and biofilm formation were downregulated upon QS inhibition. In contrast, QS stimulation led to an upregulation of the above-mentioned genes compared to the control. In summary, these results reveal the impact of QS on the activity of D. vulgaris, paving the way toward the prevention of corrosive SRB biofilm formation via QS inhibition.IMPORTANCE Sulfate-reducing bacteria (SRB) are considered key contributors to biocorrosion, particularly in saline environments. Biocorrosion imposes tremendous economic costs, and common approaches to mitigate this problem involve the use of toxic and hazardous chemicals (e.g., chlorine), which raise health and environmental safety concerns. Quorum-sensing inhibitors (QSIs) can be used as an alternative approach to inhibit biofilm formation and biocorrosion. However, this approach would only be effective if SRB rely on QS for the pathways associated with biocorrosion. These pathways would include biofilm formation, electron transfer, and metabolism. This study demonstrates the role of QS in Desulfovibrio vulgaris on the above-mentioned pathways through both phenotypic measurements and transcriptomic approach. The results of this study suggest that QSIs can be used to mitigate SRB-induced corrosion problems in ecologically sensitive areas.

Interfacial Polymerization of Zwitterionic Building Blocks for High-Flux Nanofiltration Membranes.

A simple scalable strategy is proposed to fabricate highly permeable antifouling nanofiltration membranes. Membranes with a selective thin polyamide layer were prepared via interfacial polymerization incorporating building blocks of zwitterionic copolymers. The zwitterionic copolymer, poly(aminopropyldimethylaminoethyl methacrylate)- co-poly(sulfobetaine methacrylate) with an average molecular weight of 6.1 kg mol-1, was synthesized in three steps: (i) polymerization of dimethylaminoethyl methacrylate to yield the base polymer by atom transfer radical polymerization (ATRP), (ii) fractional sulfobetainization via quaternization, and (iii) amination via quaternization. The effect of the zwitterionic polymer content on the polyamide surface characteristics, fouling resistance, and permeance is demonstrated. The zwitterion-modified membrane becomes more hydrophilic with lower surface roughness, as the zwitterionic polymer fraction increases. The excellent fouling resistance of the zwitterion-modified membrane was confirmed by the negligible protein adsorption and low bacteria fouling compared to a pristine membrane without zwitterionic segments. In addition, the zwitterion-modified membranes achieve a water permeation around 135 L m-2 h-1bar-1, which is 27-fold higher than that of the pristine membrane, along with good selectivity in the nanofiltration range, confirmed by the rejection of organic dyes. This permeance is about 10 times higher than that of other reported loose nanofiltration membranes with comparable dye rejection. The newly designed membrane is promising as a highly permeable fouling resistant cross-linked polyamide network for various water treatment applications.

Acquisition of Extracellular DNA by Acinetobacter baylyi ADP1 in Response to Solar and UV-C254nm Disinfection.

Extracellular DNA (eDNA) cannot be effectively removed by most of the existing wastewater treatment technologies and can contribute to the gain of new functional traits when transformed into competent bacteria present in downstream environments. This study evaluates the contributions of solar and UV-C254nm irradiation to the transformation of eDNA in Acinetobacter baylyi ADP1. Solar irradiation was evaluated because it is a natural environmental stressor to which eDNA would be exposed during wastewater reuse. UV-C254nm was evaluated as an alternative to a chlorine-based disinfection strategy. Our findings showed that solar disinfection increased the natural transformation frequency by up to 2.0-fold after irradiance at 153 mJ/cm2. This was largely mediated by reactive oxygen species generation, which was correlated with an upregulation of both DNA repair (recA and ddrR) and competence (comA and pilX) genes. In contrast, even though UV-C254nm exposure was accompanied by the upregulation of DNA repair (recA, ddrR, and uvrB) genes and, hence, possibly higher integration rates of eDNA, we observed a concentration-dependent decrease in transformation rates. This decrease in transformation was likely due to the UV dimerization of eDNA, which resulted in the integration of damaged genes that cannot be transcribed into any functional gene products. These results imply that even though sunlight stimulates eDNA uptake and integration in the natural environment, UV disinfection implemented at a treatment plant can potentially minimize subsequent detrimental effects by damaging the extracellular genetic material and ensuring that there is no substantial expression of these transformed genes.

Water Disinfection Byproducts Increase Natural Transformation Rates of Environmental DNA in Acinetobacter baylyi ADP1.

The process of natural transformation allows for the stable uptake, integration, and functional expression of extracellular DNA. This mechanism of horizontal gene transfer has been widely linked to the acquisition of antibiotic resistance and virulence factors. Here, we demonstrate that bromoacetic acid (BAA)-a regulated drinking water disinfection byproduct (DBP)-can stimulate natural transformation rates in the model organism Acinetobacter baylyi ADP1. We demonstrate that transformation stimulation in response to BAA is concentration-dependent and is linked to the ability of this compound to generate DNA damage via oxidative stress. In presence of BAA, transcription of recA was upregulated 20-40% compared to the nontreated controls, indicating that this component of the DNA damage response could be associated with the increase in transformation. Other genes associated with DNA translocation across the cytoplasmic membrane (i.e., pilX, comA) did not exhibit increased transcription in the presence of BAA, indicating that the enhancement of transformation is not associated with increased translocation rates of environmental DNA. Overall, these results lead us to speculate that elevated recA transcription levels could lead to increased integration rates of foreign DNA within the recipient cell during DNA repair. Lastly, we show that an artificial DBP cocktail simulating the environmental concentrations of five water DBP classes stimulates natural transformation by almost 2-fold. The results of this study suggest that mutagens like DBPs may play an important role in enhancing the fixation rates of extracellular DNA in the environmental metagenome.

Bacteriophages To Sensitize a Pathogenic New Delhi Metallo ß-Lactamase-Positive Escherichia coli to Solar Disinfection.

Bacteriophages active against a New Delhi metallo beta lactamase (NDM)-positive E. coli PI-7 were isolated from municipal wastewater and tested for their lytic effect against the bacterial host. Bacteriophages were highly specific to E. coli PI-7 when tested for host-range. After determining host-specificity, bacteriophages were tested for their ability to sensitize E. coli PI-7 to solar irradiation. Solar irradiation coupled with bacteriophages successfully reduced the length of the lag-phase for E. coli PI-7 from 4 h to 2 h in buffer solution. The reduction of lag-phase length was also observed in filtered wastewater effluent and chlorinated effluent. Previously, we found through gene expression analysis that cell wall, oxidative stress, and DNA repair functions played a large role in protecting E. coli PI-7 against solar damage. Here, gene expression analysis of bacteriophage-supplemented solar-irradiated E. coli PI-7 revealed downregulation of cell wall functions. Downregulation of functions implicated in scavenging and detoxifying reactive oxygen species, as well as DNA repair genes, was also observed in bacteriophage-supplemented solar-irradiated E. coli PI-7. Moreover, solar irradiation activates recA, which can induce lytic activity of bacteriophages. Overall, the combined treatment led to gene responses that appeared to make E. coli PI-7 more susceptible to solar disinfection and bacteriophage infection. Our findings suggest that bacteriophages show good potential to be used as a biocontrol tool to complement solar irradiation in mitigating the persistence of antibiotic-resistant bacteria in reuse waters.

Fate and Persistence of a Pathogenic NDM-1-Positive Escherichia coli Strain in Anaerobic and Aerobic Sludge Microcosms.

The presence of emerging biological pollutants in treated wastewater effluents has gained attention due to increased interest in water reuse. To evaluate the effectiveness of the removal of such contaminants by the conventional wastewater treatment process, the fate and decay kinetics of NDM-1-positive Escherichia coli strain PI7 and its plasmid-encoded antibiotic resistance genes (ARGs) were assessed in microcosms of anaerobic and aerobic sludge. Results showed that E. coli PI7 decayed at a significantly lower rate under anaerobic conditions. Approximate half-lives were 32.4 ± 1.4 h and 5.9 ± 0.9 h in the anaerobic and aerobic microcosms, respectively. In the aerobic microcosms, after 72 h of operation, E. coli PI7 remained detectable, but no further decay was observed. Instead, 1 in every 10,000 E. coli cells was identified to be recalcitrant to decay and persist indefinitely in the sludge. ARGs associated with the E. coli PI7 strain were detected to have transferred to other native microorganisms in the sludge or were released to the liquid fraction upon host decay. Extracellular DNA quickly degraded in the liquid fraction of the aerobic sludge. In contrast, no DNA decay was detected in the anaerobic sludge water matrix throughout the 24-h sampling period. This study suggests an increased likelihood of environmental dispersion of ARGs associated with anaerobically treated wastewater effluents and highlights the potential importance of persister cells in the dissemination of E. coli in the environment during reuse events of treated wastewater.IMPORTANCE This study examines the decay kinetics of a pathogenic and antibiotic resistant strain of Escherichia coli in microcosms simulating biological treatment units of aerobic and anaerobic sludge. The results of this study point at a significantly prolonged persistence of the E. coli and the associated antibiotic resistance gene in the anaerobic sludge. However, horizontal transfer of the plasmid encoding the antibiotic resistance gene was detected in the aerobic sludge by a cultivation method. A subpopulation of persister E. coli cells was also detected in the aerobic sludge. The findings of this study suggest potential areas of concern arising from pathogenic and antibiotic-resistant E. coli during both anaerobic and aerobic sludge treatment processes.