Removal of intI1, ARGs, and SARS-CoV-2 and changes in bacterial communities in four sewage treatment facilities.

Currently, discharge regulations for wastewater treatment plants (WWTPs) are based on conventional parameters, but more is needed to ensure safe water reuse. In particular, emerging pollutants, as antimicrobials and antibiotic resistance genes (ARGs), are not considered. This research focuses on the fate of emerging biological contaminants during wastewater treatment in Mexico City. intI1 and the ARGs cphA-02, OXA-10 and sul1 were analyzed by qPCR; pathogenic bacteria species were characterized by high throughput sequencing of complete 16S rRNA gene, and fragments of SARS-CoV-2 were quantified by RT-qPCR. Conventional parameters (chemical oxygen demand and coliform bacteria) were also determined. Two sampling campaigns (rainy and dry seasons) were carried out in four municipal WWTPs in Mexico City, representing five biological treatment processes: conventional activated sludge, extended aeration activated sludge, membrane bioreactor, direct anaerobic digestion, and constructed wetland, followed by ultraviolet light or chlorine disinfection. In most cases, gene fragments of SARS-CoV-2 were eliminated below the detection limit of RT-qPCR. The abundance of intI1 positively correlated with the sul1, OXA-10, and cphA-02 abundances; intI1 and the ARGs here studied were partially removed in the WWTPs, and in most cases, the number of copies per second discarded in the sludge were higher those in the effluent. The treatment processes decreased the abundance of dominant bacterial groups in the raw wastewater, while enriching bacterial groups in the effluent and the biological sludge, with possible pollutant removal capabilities. Bacterial communities in the raw wastewater showed the predominance of the genus Arcobacter (from 62.4 to 86.0 %) containing potentially pathogenic species. Additionally, DNA of some species persisted after the treatment processes: A. johnsonii, A. junii, A. caviae, A. hydrophila, A. veronii, A. butzleri, A. cryaerophilus, Chryseobacterium indologenes, Hafnia paralvei, M. osloensis, Pseudomonas putida and Vibrio cholerae, which deserves special attention in future regulation for safe water reuse.

Removal of bacterial pathogens and antibiotic resistance bacteria by anaerobic sludge digestion with thermal hydrolysis pre-treatment and alkaline stabilization post-treatment.

Primary sludge (PS) is associated with public health and environmental risks, so regulations focus on reducing the pathogenic and heavy metal contents of the treated material (biosolids), intended for soil amendments and land reclamation. The regulations set limits for Escherichia coli (or fecal coliforms), Salmonella spp., helminth eggs and enterovirus. However, the potential risk due to antibiotic resistant bacteria (ARB) and other human potential pathogenic bacteria (HPB) are not considered. In this work, three sludge treatment processes, having in common an anaerobic digestion step, were applied to assess the removal of regulated bacteria (fecal coliforms, Salmonella spp), ARB and HPB. The treatment arrangements, fed with PS from a full-scale wastewater treatment plant were: 1) Mesophilic anaerobic digestion followed by alkaline stabilization post-treatment (MAD-CaO); 2) Thermophilic anaerobic digestion (TAD) and, 3) Pre-treatment (mild thermo-hydrolysis) followed by TAD (PT-TAD). The results address the identification, quantification (colony forming units) and taxonomic characterization of ARB resistant to ß-lactams and vancomycin, as well as the taxonomic characterization of HPB by sequencing with PacBio. In addition, quantification based on culture media of fecal coliforms and Salmonella spp. is presented. The capabilities and limitations of microbiological and metataxonomomic analyses based on PacBio sequencing are discussed, emphasizing that they complement each other. Genus Aeromonas, Acinetobacter, Citrobacter, Enterobacter, Escherichia, Klebsiella, Ochrobactrum, Pseudomonas and Raoultella, among others, were found in the PS, which are of clinical or environmental importance, being either HPB, HPB-ARB, or non-pathogenic ARB with the potentiality of horizontal gene transfer. Based on the analysis of fecal coliforms and Salmonella spp., the three processes produced class A (highest) biosolids, suitable for unrestricted agriculture applications. Mild thermo-hydrolisis was effective in decreasing ARB cultivability, but it reappeared after the following TAD. O. intermedium (HPB-ARB) was enriched in MAD and TAD while Laribacter hongkongensis (HPB) did persist after the applied treatments.

Next-generation Sequencing for Surveillance of Antimicrobial Resistance and Pathogenicity in Municipal Wastewater Treatment Plants.

The World Health Organization (WHO) ranks antimicrobial resistance (AMR) and various pathogens among the top 10 health threats. It is estimated that by 2050, the number of human deaths due to AMR will reach 10 million annually. On the other hand, several infectious outbreaks such as SARS, H1N1 influenza, Ebola, Zika fever, and COVID-19 have severely affected human populations worldwide in the last 20 years. These recent global diseases have generated the need to monitor outbreaks of pathogens and AMR to establish effective public health strategies. This review presents AMR and pathogenicity associated with wastewater treatment plants (WWTP), focusing on Next Generation Sequencing (NGS) monitoring as a complementary system to clinical surveillance. In this regard, WWTP may be monitored at three main points. First, at the inlet (raw wastewater or influent) to identify a broad spectrum of AMR and pathogens contained in the excretions of residents served by sewer networks, with a specific spatio-temporal location. Second, at the effluent, to ensure the elimination of AMR and pathogens in the treated water, considering the rising demand for safe wastewater reuse. Third, in sewage sludge or biosolids, their beneficial use or final disposal can represent a significant risk to public health. This review is divided into two sections to address the importance and implications of AMR and pathogen surveillance in wastewater and WWTP, based on NGS. The first section presents the fundamentals of surveillance techniques applied in WWTP (metataxonomics, metagenomics, functional metagenomics, metaviromics, and metatranscriptomics). Their scope and limitations are analyzed to show how microbiological and qPCR techniques complement NGS surveillance, overcoming its limitations. The second section discusses the contribution of 36 NGS research papers on WWTP surveillance, highlighting the current situation and perspectives. In both sections, research challenges and opportunities are presented.

Performance evaluation and kinetic modeling of an upflow anaerobic sludge blanket septic tank for domestic wastewater treatment.

This work evaluated the UASB-septic tank performance using different kinetic models that correlated process efficiency and methane production with hydraulic and organic loading rates through experiments with five different HRT (48 h, 36 h, 24 h, 18 h, and 12 h) using synthetic domestic wastewater. The modified Stover-Kincannon model provided the best fitting to calculate kinetics constants, with an R2 above 98% for linear regression, and predicted the effluent COD more accurately than the other models. Methane yield was 0.3294 L CH4/g COD removed, being closer to the theoretical value, and the Van der Meer and Heertjes model had the highest R2 for methane production. Organic matter and solids removal were 45% for TS, 70% and 68% for total and soluble COD, and 85% for TSS. Pollutant removal markedly decreased when the reactor operated HRT below 24 h; thus, it is recommended to operate the UASB-septic tank at this HRT.

Photorespiration in an outdoor alkaline open-photobioreactor used for biogas upgrading.

The rates of oxygenic and carbon fixation photosynthetic processes of a microalgae consortium were simultaneously evaluated under steady-state performance in an bench scale alkaline open-system exposed to outdoor conditions in Mexico City. A synthetic methane-free gaseous stream (SMGS) similar to biogas was used as inorganic carbon source and model of biogas upgrading. The microalgae CO2 fixation rates were calculated through a novel methodology based on an inorganic carbon mass balance under continuous scrubbing of a SMGS similar to biogas, where the influence of pH and temperature time-depended oscillations were successfully incorporated into the mass balances. The oxygenic activity and carbon fixation occurred at different non-stoichiometric rates during the diurnal phase, in average carbon fixation predominated over oxygen production (photosynthesis quotient PQ≈ 0.5 mol O2 mol-1 CO2) indicating photorespiration occurrence mainly under dissolved oxygen concentrations higher than 10 mg L-1. The oxygen and inorganic carbon mass balances demonstrated that photorespiration and endogenous respiration were responsible for losing up to 66% and 7% respectively of the biomass grew at diurnal periods under optimal conditions. In favoring photorespiration conditions, the microalgae biomass productivity (CO2 effectively captured) can be severely decreased. A kinetic mathematical model as a function of temperature and irradiance of the oxygenic photosynthetic activity indicated the optimal operation zone for this outdoor alkaline open-photobioreactor, where irradiance was found being the most influential parameter.

Aerobic biodegradation of a sulfonated phenylazonaphthol dye by a bacterial community immobilized in a multistage packed-bed BAC reactor.

A microbial community able to aerobically degrade the azo dye Acid Orange 7 was selected from riparian or lacustrine sediments collected at sites receiving textile wastewaters. Three bacterial strains, pertaining to the genera Pseudomonas, Arthrobacter, and Rhizobium, constitute the selected community. The biodegradation of AO7 was carried out in batch-suspended cell culture and in a continuously operated multistage packed-bed BAC reactor. The rapid decolorization observed in batch culture, joined to a delay of about 24 h in COD removal and cell growth, suggests that enzymes involved in biodegradation of the aromatic amines generated after AO7 azo-bond cleavage (1-amino-2-naphthol [1-A2N] and 4-aminobenzenesulfonic acid [4-ABS]), are inducible in this microbial consortium. After this presumptive induction period, the accumulated byproducts, measured through COD, were partially metabolized and transformed in cell mass. At all azo dye loading rates used, complete removal of AO7 and 1-A2N was obtained in the multistage packed-bed BAC reactor (PBR).; however, the overall COD (eta ( COD )) and 4-ABS (eta ( ABS )) removal efficiencies obtained in steady state continuous culture were about 90%. Considering the toxicity of 1-A2N, its complete removal has particular relevance. In the first stages of the packed-bed BAC reactor (Fig. 4a-c), major removal was observed. In the last stage, only a slight removal of COD and 4-ABS was obtained. Comparing to several reported studies, the continuously operated multistage packed-bed BAC reactor showed similar or superior results. In addition, the operation of large-packed-bed BAC reactors could be improved by using several shallow BAC bed stages, because the pressure drop caused by bed compaction of a support material constituted by small and fragile particles can be reduced.