Artificial Sweetener Acesulfame Research Articles

A Novel Sulfatase for Acesulfame Degradation in Wastewater Treatment Plants as Evidenced from Shinella Strains.

The artificial sweetener acesulfame is a persistent pollutant in wastewater worldwide. So far, only a few bacterial isolates were recently found to degrade acesulfame efficiently. In Bosea and Chelatococcus strains, a Mn2+-dependent metallo-β-lactamase-type sulfatase and an amidase signature family enzyme catalyze acesulfame hydrolysis via acetoacetamide-N-sulfonate to acetoacetate. Here, we describe a new acesulfame sulfatase in Shinella strains isolated from wastewater treatment plants in Germany. Their genomes do not encode the Mn2+-dependent sulfatase. Instead, a formylglycine-dependent sulfatase gene was found, together with the acetoacetamide-N-sulfonate amidase gene on a plasmid shared by all known acesulfame-degrading Shinella strains. Heterologous expression, proteomics, and size exclusion chromatography corroborated the physiological function of the Shinella sulfatase in acesulfame hydrolysis. Since both acesulfame sulfatase types are absent in other bacterial genomes or metagenome-assembled genomes, we surveyed 73 tera base pairs of wastewater-associated metagenome raw data sets. Bosea/Chelatococcus sulfatase gene signatures were regularly found from 2013, particularly in North America, Europe, and East Asia, whereas Shinella sulfatase gene signatures were first detected in 2020. Moreover, signatures for the Shinella sulfatase and amidase genes co-occur only in six data sets from China, Finland, and Mexico, suggesting that the Shinella genes were enriched or introduced quite recently in wastewater treatment facilities.

Influence of vegetation and substrate type on removal of emerging organic contaminants and microbial dynamics in horizontal subsurface constructed wetlands

Constructed wetlands (CWs) offer an efficient alternative technology for removing emerging organic contaminants (EOCs) from wastewater. Optimizing CW performance requires understanding the impact of CW configuration on EOC removal and microbial community dynamics. This study investigated EOC removal and microbial communities in horizontal subsurface flow (HSSF) CWs over a 26-month operational period. Comparison between tuff-filled and gravel-filled CWs highlighted the superior EOC removal in tuff-filled CWs during extended operation, likely caused by the larger surface area of the tuff substrate fostering microbial growth, sorption, and biodegradation. Removal of partially positively charged EOCs, like atenolol (29–98 %) and fexofenadine (21–87 %), remained constant in the different CWs, and was mainly attributed to sorption. In contrast, removal rates for polar non-sorbing compounds, including diclofenac (3–64 %), acyclovir (9–85 %), and artificial sweeteners acesulfame (5–60 %) and saccharin (1–48 %), seemed to increase over time due to enhanced biodegradation. The presence of vegetation and different planting methods (single vs. mixed plantation) had a limited impact, underscoring the dominance of substrate type in the CW performance. Microbial community analysis identified two stages: a startup phase (1–7 months) and a maturation phase (19–26 months). During this transition, highly diverse communities dominated by specific species in the early stages gave way to more evenly distributed and relatively stable communities. Proteobacteria and Bacteroidetes remained dominant throughout. Alphaproteobacteria, Acidobacteria, Planctomycetes, Salinimicrobium, and Sphingomonas were enriched during the maturation phase, potentially serving as bioindicators for EOC removal. In conclusion, this study emphasizes the pivotal role of substrate type and maturation in the removal of EOCs in HSSF CW, considering the complex interplay with EOC physicochemical properties. Insights into microbial community dynamics underscore the importance of taxonomic and functional diversity in assessing CW effectiveness. This knowledge aids in optimizing HSSF CWs for sustainable wastewater treatment, EOC removal, and ecological risk assessment, ultimately contributing to environmental protection.

Acesulfame degradation by thermally activated persulfate: Kinetics, transformation products and estimated toxicity

The existence of the artificial sweetener acesulfame (ACE) in quantities of significance can negatively impact water quality, and its consumption has been associated with deleterious health effects. The present investigation explores the efficacy of heat-activated sodium persulfate (SPS) for eliminating ACE. The complete degradation of 0.50 mg L−1 of ACE was achieved within 45 min under a reaction temperature of 50 °C and 100 mg L−1 of SPS. The impact of thermal decomposition on ACE at a temperature of 60 °C was negligible. This study considers several factors, such as the SPS and ACE loading, the reaction temperature, the initial pH, and the water matrix of the reactor. The results indicate that the method's efficiency is positively correlated with higher initial concentrations of SPS, whereas it is inversely associated with the initial concentration of ACE. Furthermore, higher reaction temperatures and acidic initial pH levels promote the degradation of acesulfame. At the same time, certain constituents of the water matrix, such as humic acid, chlorides, and bicarbonates, can hinder the degradation process. Additionally, the data from LC-QToF-MS analysis of the samples were used to investigate transformation through suspect and non-target screening approaches. Overall, ACE's eight transformation products (TPs) were detected, and a potential ACE decomposition pathway was proposed. The concentration of TPs followed a volcano curve, decreasing in long treatment times. The ecotoxicity of ACE and its identified TPs was predicted using the ECOSAR software. The majority of TPs exhibited not harmful values.

Simultaneous analysis of natural and artificial sweeteners in sugar-free drinks and urine samples by column-switching UHPLC-charged aerosol detection method

Sweeteners are considered an alternative to high-calorie foods or drinks and have been widely used globally. However, the simultaneous separation and detection of high-polarity natural and artificial sweeteners are challenging owing to their broad-spectrum physical and chemical properties. Herein, we developed a column-switching UHPLCCAD method and used it for detecting and quantitating 12 sweeteners, including natural sweeteners (erythritol, mannitol, xylitol, sorbitol and stevioside) and artificial sweeteners (acesulfame potassium, saccharin sodium salt, sodium cyclamate, sucralose, aspartame, alitame and neotame). The LOD and LOQ were 0.932–6.25 μg/mL and 3.10–20.83 μg/mL, respectively, and the method demonstrated excellent linearity (R² ≥ 0.9990), good precision (intraday and interday precision was 0.59–6.88 %), and high recovery (average recoveries were 85.16–108.64 %). This method was applied to determine the sweeteners in 15 sugar-free drinks purchased from the local Chinese supermarkets. What's more, natural sweetener erythritol and artificial sweetener acesulfame potassium were suspected over addition in sugar-free drinks. Meanwhile the method was applied to the sweeteners in various sugar-free drinks and the dynamic monitoring of transit and excretion in vivo after drinking. Those prove that the method can be used to the detection of sugar free drinks and quality control of the sweeteners. The study highlights the potential of UHPLC–charged aerosol detection technology in detection of multiple components in food industry.

Transport and Attenuation of an Artificial Sweetener and Six Pharmaceutical Compounds in a Sequenced Wetland-Steel Slag Wastewater Treatment System

The occurrence of pharmaceutically active compounds (PhACs), nutrients, and an artificial sweetener acesulfame in wastewater, and subsequent removal in an engineered system comprising aerobic wetland, anaerobic wetland, and steel slag cells, were investigated. The PhACs evaluated in this study covered a range of octanol–water partition coefficients (log Kow = 0.07–2.45) and acid dissociation constants (pKa = 1.7–13.9) and included carbamazepine, caffeine, sulfamethoxazole, ibuprofen, and naproxen. The mean flow rate in the system was 0.89 m3 day−1 (0.02 to 4.27 m3 day−1), representing a hydraulic retention time of 5 days. The removal efficiencies of PO4-P, NH3-N, and cBOD5 in the treatment system were >99, 82, and 98%. The removal efficiencies for the PhACs and acesulfame were classified into four groups, including those that were (a) efficiently removed (caffeine by >75%); (b) moderately removed (ibuprofen by 50–75%); (c) poorly removed (sulfamethoxazole and naproxen by 25–50%); and (d) recalcitrant (carbamazepine and acesulfame by <25%). Variability in concentrations and treatment efficiencies was observed in different sampling events, which may be due to variations in input concentrations or changes in the flow rate. The addition of a steel slag cell increased the overall removal efficiency of the studied compounds, except for carbamazepine.

Sonochemical degradation of the artificial sweetener acesulfame in aqueous medium and identification of transformation products

The sonochemical degradation of acesulfame (ACE), a widely used artificial sweetener, was investigated. The impact of power density (40 and 71 W/L), initial ACE concentration (1.5–30.6 μΜ), and initial solution pH (3–9) for the elimination of the target pollutant from water was studied. It was found that nearly complete degradation of ACE was achieved within 120 min of treatment under conditions suitable for high production of free radicals. Degradation follows a pseudo-first order kinetic model. Coupling sonochemical degradation at optimal conditions with sodium persulfate (0.2–1.0 mM) enhanced the rate of ACE oxidation by six times. The influence of water complexity was also examined; the natural occurrence of humic acid in the water matrix reduced the degradation rate, despite the fact that at least 42 % of ACE removal was achieved. Finally, LC-QToF-MS was used to evaluate the generated transformation products (TPs). Using HRMS suspect and non-target screening, seven ACE TPs were identified, and their time-trend formation profiles showed a rising trend throughout the experiment. According to the ECOSAR model, most of the identified transformation products exhibited lower toxicity than acesulfame, with one notable exception that was classified as harmful due to its acute toxicity on fish and green algae.

Domesticating aquatic plants in hydroponic systems to demonstrate and advance phytoremediation of the artificial sweetener acesulfame

Artificial sweeteners (ASs) are ubiquitously detected in the global water system, among which acesulfame (ACE) is an emerging contaminant for its chemical and biological stability and unsatisfying removal by conventional or advanced treatment technologies. Phytoremediation is an effective and sustainable in-situ remediation technology that this study is the first to explore ACE removal by aquatic plants. The emergent plants, Scirpus Validus (S. validus), Phyllostachys heteroclada Oliver (P. heteroclada) and Acorus tatarinowii (A. tatarinowii) showed superior removal capability than eleven floating plants, and demonstrated high phytoremediation efficiencies (PEs) of up to 75 % after 28 d domestication. ACE removal by the three emergent plants increased during domestication, as the PEs after 28 d domestication were 5.6–6.5 times of 7 d domestication. Notably, the half-life of ACE was decreased from 20.0 to 33.1 d to 1.1–3.4 d in the plant-hydroponic system, compared with 481.0–1152.4 d in control water without plants. Moreover, A. tatarinowii demonstrated the highest removal capacity for ACE with 0.37 mg/g fresh biomass weight (FW), higher than S. validus (0.27 mg/g FW) and P. heteroclada (0.20 mg/g FW). It is worth noting that a mass balance analysis demonstrated that plant transpiration and plant uptake account for about 6.72 %–18.54 % and 9.69 %–21.67 % ACE removal, while hydrolysis only accounted for about 4 % and photolysis was negligible. The rest ACE may be used as a carbon source by endophytic bacteria and root microorganisms of plants. In addition, increased temperature, pH, and illumination intensity had a significant effect on phytoremediation. In the selected experimental range, the increase of temperature from 15 °C to 35 °C, illumination intensity from 1500 lx to 6000 lx, and pH from 5 to 9 generally accelerated the PEs of ACE during the domestication process. Though the mechanism still requires further investigation, the results provide scientific and feasible data for removal of ACE from water by diverse plants for the first time, and also revealed insights for in-situ treatment of ACE.

Seasonal biodegradation of the artificial sweetener acesulfame enhances its use as a transient wastewater tracer

The persistence of the artificial sweetener acesulfame potassium (ACE) during wastewater treatment and subsequently in the aquatic environment has made it a widely used tracer of wastewater inputs to both surface water and groundwater. However, the recently observed biodegradation of ACE during wastewater treatment has questioned the validity of this application. In this study, we assessed the use of ACE not only as a marker of wastewater, but also as a transient wastewater tracer that allows both the calculation of mixing ratios and travel times through the aquifer as well as the calibration of transient groundwater flow and mass transport models. Our analysis was based on data obtained in a nearly 8-year river water and groundwater sampling campaign along a confirmed wastewater-receiving riverbank filtration site located close to a drinking water supply system. We provide evidence that temperature controls ACE concentration and thus its seasonal oscillation. River water data showed that ACE loads decreased from 1.5–4 mg·s−1 in the cold season (December to June; T<10 °C) to 0–0.5 mg·s−1 in the warm season (July to November; T>10 °C). This seasonal variability of >600% was detectable in the aquifer and preserved >3 km, with ACE concentrations oscillating between <LOQ in the warm season up to 1 μg·L−1 in the cold season. The large seasonal variation in ACE concentrations during wastewater treatment, compared to the other sweeteners (sucralose, cyclamate, and saccharin) and chloride enables its use as a transient tracer of wastewater inflows and riverbank filtration. In addition, the arrival time of the ACE concentration peak can be used to estimate groundwater flow velocity and mixing ratios, thereby demonstrating its potential in the calibration of groundwater numerical models.

Protein fractionation and shotgun proteomics analysis of enriched bacterial cultures shed new light on the enzymatically catalyzed degradation of acesulfame

The removal of organic micropollutants in municipal wastewater treatment is an extensively studied field of research, but the underlying enzymatic processes have only been elucidated to a small extent so far. In order to shed more light on the enzymatic degradation of the artificial sweetener acesulfame (ACE) in this context, we enriched two bacterial taxa which were not yet described to be involved in the degradation of ACE, an unknown Chelatococcus species and Ensifer adhaerens, by incubating activated sludge in chemically defined media containing ACE as sole carbon source. Cell-free lysates were extracted, spiked with ACE and analyzed via target LC-MS/MS, demonstrating for the first time enzymatically catalyzed ACE degradation outside of living cells. Fractionation of the lysate via two-dimensional fast protein liquid chromatography (FPLC) succeeded in a partial separation of the enzymes catalyzing the initial transformation reaction of ACE from those catalyzing the further transformation pathway. Thereby, an accumulation of the intermediate transformation product acetoacetamide-n-sulfonic acid (ANSA) in the ACE-degrading fractions was achieved, providing first quantitative evidence that the cleavage of the sulfuric ester moiety of ACE is the initial transformation step. The metaproteome of the enrichments was analyzed in the FPLC fractions and in the unfractionated lysate, using shotgun proteomics via UHPLC-HRMS/MS and label-free quantification. The comparison of protein abundances in the FPLC fractions to the corresponding ACE degradation rates revealed a metallo-β-lactamase fold metallo-hydrolase as most probable candidate for the enzyme catalyzing the initial transformation from ACE to ANSA. This enzyme was by far the most abundant of all detected proteins and amounted to a relative protein abundance of 91% in the most active fraction after the second fractionation step. Moreover, the analysis of the unfractionated lysate resulted in a list of further proteins possibly involved in the transformation of ACE, most striking a highly abundant amidase likely catalyzing the further transformation of ANSA, and an ABC transporter substrate-binding protein that may be involved in the uptake of ACE into the cell.

Innovation versus novelty in microbial systems.

Innovation versus novelty in microbial systems.

Back Cover: Consumption of Artificial Sweetener Acesulfame Potassium Increases Preterm Risk and Uterine Contraction with Calcium Influx Increased via Myosin Light Chain Kinase–Myosin Light Chain 20 Related Signaling Pathway

Mol. Nutr. Food Res. 2022, 66, 202200298 DOI: 10.1002/mnfr.202200298 The usage of non-nutrition sweeteners (NNS) is rapidly increasing in the food industry, with Acesulfame Potassium (Ace K) as the most prevalently consumed NNS in soft drinks. Exposure to Ace K increases the preterm risk, cytokine secretion and uterine hypercontraction through the modulation of calcium influx and alters MLCK/MLC20 contraction related protein expression through c-Jun N-terminal kinase signaling transduction. This is reported by Yang-Ching Chen, Shih-Min Hsia, and co-workers in article number 2200298.

Consumption of Artificial Sweetener Acesulfame Potassium Increases Preterm Risk and Uterine Contraction with Calcium Influx Increased via Myosin Light Chain Kinase–Myosin Light Chain 20 Related Signaling Pathway

The consumption of artificial sweeteners has been rapidly increasing, with potentially hazardous effects on human reproduction. This study aims to explore the effect of Acesulfame Potassium (Ace K) and its potential mechanism to induce uterine contraction through in vitro, ex vivo, in vivo, and clinical observation studies. Used ex vivo and in vitro studies to analyze its effect on uterine contraction and involved signaling pathway. Used the long-term, high-dose exposure to examine Ace K's affection for contractive-related protein expression. By involving a cohort of 613 participants, to assess the dose-responsiveness of Ace K consumption and calculate the odd ratio of Ace K consumption and the relationship with preterm risk. Animal studies show increasing uterine contraction, cytokine secretion, and altered contraction-related protein expression. Human data show that higher consumption of Ace K may be related to early delivery. Long-term high-dose exposure to Ace K can induce uterine hypercontraction, increase cytokine secretion, and alters contraction-related protein expression. These findings suggest that women who suffer from uterine hypercontraction causes painfulness should pay more attention to the zero- or low-calorie soft drinks or food products containing Ace K.

Comparative efficiency of oxidation processes to remove acesulfame in water treatment plants supplied by surface water sources

Abstract The effects of oxidizing agents during water treatment on the concentration of an artificial sweetener were evaluated in full-scale conditions. Five drinking water treatment plants (DWTPs) located in a northern environment with high seasonal variations which use different raw water sources and different combinations of oxidants (ozone, chlorine, UV radiation) were investigated through the removal of the artificial sweetener acesulfame (ACE) along their treatment chains. In total, 98 sampling campaigns were conducted at these DWTPs. Raw water (impacted by variable tidal and hydrodynamic conditions), partially treated water within the DWTPs, and fully treated drinking water were sampled during eight months over the period of higher variability of source water quality. Results showed ACE concentrations in raw waters vary on a seasonal basis: higher in winter and summer (when rivers have low water discharges) and lower during spring and fall. Multi-barrier treatment systems under study were effective for the removal of acesulfame due specifically to the effect of ozone and chlorine during oxidation steps, while no removal was observed using physico-chemical (coagulation flocculation, filtration) and UV treatments. Depending on the number of treatment steps that involved ozonation or chlorination and the position of these oxidative processes in the treatment chain, removal of ACE varied from 24% to 90% in the plants under study. The results indicate that increasing oxidant doses would result in better removal of ACE and other contaminants, but these strategies must consider unknown transformation products, potentially with greater toxicological effects than their precursors.

RNA in Municipal Wastewater Reveals Magnitudes of COVID-19 Outbreaks across Four Waves Driven by SARS-CoV-2 Variants of Concern.

There are no standardized protocols for quantifying severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in wastewater to date, especially for population normalization. Here, a pipeline was developed, applied, and assessed to quantify SARS-CoV-2 and key variants of concern (VOCs) RNA in wastewater at Saskatoon, Canada. Normalization approaches using recovery ratio and extraction efficiency, wastewater parameters, or population indicators were assessed by comparing to daily numbers of new cases. Viral load was positively correlated with daily new cases reported in the sewershed. Wastewater surveillance (WS) had a lead time of approximately 7 days, which indicated surges in the number of new cases. WS revealed the variant α and δ driving the third and fourth wave, respectively. The adjustment with the recovery ratio and extraction efficiency improved the correlation between viral load and daily new cases. Normalization of viral concentration to concentrations of the artificial sweetener acesulfame K improved the trend of viral load during the Christmas and New Year holidays when populations were dynamic and variable. Acesulfame K performed better than pepper mild mottle virus, creatinine, and ammonia for population normalization. Hence, quality controls to characterize recovery ratios and extraction efficiencies and population normalization with acesulfame are promising for precise WS programs supporting decision-making in public health.

Septic System Impacts on Source Water: Two Novel Field Tracer Experiments in Fractured Sedimentary Bedrock

Septic systems are a common contributor of contaminants to groundwater that have implications for source water protection, particularly in fractured sedimentary bedrock environments. Two 24-h tracer experiments were performed that applied (1) the dye Lissamine Flavine FF and (2) three artificial sweeteners (acesulfame, sucralose, and cyclamate) in the leaching bed to examine solute transport from a single-family septic bed to a multilevel monitoring well installed in fractured sedimentary bedrock on a First Nation reserve in Southern Ontario, Canada. Tracer was first observed 3 h and 20 min after deployment, and breakthrough curves showed that multiple pathways likely exist between the septic bed and the monitoring well. Cyclamate concentrations were more elevated than expected compared to other studies that examined cyclamate’s attenuation in the laboratory and in porous media aquifers. Solute transport through the septic bed was analyzed with the numerical modeling software Hydrus 1D, which indicated that the septic bed may be too thin, located directly on bedrock, underlain by fractured soils, or bypassed through a short-circuit. The rapid transport of septic leachate to fractured sedimentary aquifers is problematic for First Nation and rural communities. More stringent regulations are needed for the design and use of septic systems in these environments.

Variation in septic system effluent inputs to tributaries in multiple subwatersheds and approaches to distinguish contributing pathways and areas

Quantifying the contribution of septic systems to contaminant, including nutrient, loading to streams is needed in many watersheds to inform water quality management programs. However, this quantification is challenging due to the distributed locations of septic systems and uncertainties regarding the pathways delivering effluent from septic systems (functioning and failing) to a stream. The objectives of this study were firstly to evaluate how septic effluent inputs to streams vary with stream discharge conditions for multiple subwatersheds with different characteristics (i.e., geology, septic system density, and typical age), and secondly to examine new approaches for distinguishing the pathways and the contributing areas delivering septic effluent to streams. These approaches use the artificial sweetener acesulfame as a conservative tracer for septic effluent in applications of: (i) stream concentration-discharge (C-Q) relationships using low frequency sampling data, (ii) hysteresis behavior in event-based C-Q relationships, and (iii) longitudinal stream sampling. For all nine subwatersheds studied, the amount of septic effluent reaching the subwatershed outlets was considerably higher during high stream discharge (event) conditions compared to low discharge (baseflow) conditions, suggesting pathways other than groundwater may also be important. Generally, the percentage of septic effluent reaching the outlets was less for subwatersheds with newer households compared to those with older households. The combined interpretation of low frequency and event-based C-Q relationships indicate that complex pathways control the delivery of septic effluent to the subwatershed outlets. The interpretations suggest that groundwater pathways may dominate in some subwatersheds, while more rapid pathways associated with failing septic systems (e.g., overland runoff) may be important in others. Finally, longitudinal stream sampling illustrate the potential of acesulfame data to identify key areas contributing septic effluent to the stream. The novel approaches used here can be applied to guide future investigations aiming to quantify and manage water quality impairment from septic systems.

Acesulfame potassium induces dysbiosis and intestinal injury with enhanced lymphocyte migration to intestinal mucosa.

The artificial sweetener acesulfame potassium (ACK) is officially approved as safe for intake and has been used in processed foods. However, ACKs have been reported to induce metabolic syndrome, along with alteration of the gut microbiota in mice. In recent years, studies have suggested that this artificial sweetener promotes myeloperoxidase reactivity in Crohn's disease-like ileitis. We aimed to investigate the effect of ACK on the intestinal mucosa and gut microbiota of normal mice. Acesulfame potassium was administered to C57BL/6J mice (8weeks old) via free drinking. Intestinal damage was evaluated histologically, and messenger RNA (mRNA) levels of TNF-α, IFN-γ, IL1-β, MAdCAM-1, GLP1R, and GLP2R were determined with quantitative reverse transcription polymerase chain reaction (qRT-PCR). Immunohistochemistry was performed to examine the expression of MAdCAM-1 in the small intestine. The composition of gut microbiota was assessed using high-throughput sequencing. We performed intravital microscopic observation to examine if ACK altered lymphocyte migration to the intestinal microvessels. Acesulfame potassium increased the expression of proinflammatory cytokines, decreased the expression of GLP-1R and GLP-2R, and induced small intestinal injury with an increase in intestinal permeability, and ACK treatment induced microbial changes, but the transfer of feces alone from ACK mice did not reproduce intestinal damage in recipient mice. ACK treatment significantly increased the migration of lymphocytes to intestinal microvessels. Acesulfame potassium induces dysbiosis and intestinal injury with enhanced lymphocyte migration to intestinal mucosa. Massive use of non-caloric artificial sweeteners may not be as safe as we think.

Enhancement of antibiotic resistance dissemination by artificial sweetener acesulfame potassium: Insights from cell membrane, enzyme, energy supply and transcriptomics

The abuse of antibiotics on animals could induce the development of antibiotic resistant genes (ARGs) and antibiotic resistant bacteria (ARB), and acesulfame potassium (ACE) is the widely used artificial sweetener in animal feed. Generally speaking, ACE and ARB often coexist in livestock wastewater, however, the impact of the co-occurrence of ACE and ARB on the transmission of ARGs is still unknown. In this study, the effects of ACE on vertical gene transfer (VGT) and horizontal gene transfer (HGT) were both evaluated. For VGT, ACE may hinder the spread of sul gene in Pseudomonas HLS-6 by blocking ARB growth. As for HGT (from Escherichia coli DH5α to Pseudomonas HLS-6), environmentally relevant ACE concentration could facilitate the conjugative transfer. The underlying mechanisms of HGT were characterized by enhanced cell membrane permeability, reactive oxygen species overproduction, SOS response, energy supply, which were all further verified by the changes in transcription levels of related genes. Interestingly, intracellular Mg2+ in donor strain was found for the first time as an indicator for the conjugation occurrence in ACE treated mating system. This study may provide new insights into the role of ACE on ARGs proliferation and highlight its potential environmental impacts.

Acesulfame aerobic biodegradation by enriched consortia and Chelatococcus spp.: Kinetics, transformation products, and genomic characterization

The artificial sweetener Acesulfame (ACE) has been frequently detected in wastewater treatment plants (WWTPs) and is regarded as an emerging pollutant due to its low biodegradability. However, recent observations of ACE biodegradation in WWTPs have stimulated interest in the ACE-degrading bacteria and mineralization pathways. In this study, next-generation sequencing methods, Illumina and Nanopore sequencing, were combined to explore the ACE-degrading communities enriched from the activated sludge of six municipal wastewater treatment plants. Metagenomic investigations indicated that all enrichments were similarly dominated by the phyla Proteobacteria and Planctomycetes. Notably, at the species level, four metagenome-assembled genomes (MAGs) were shared by six enriched communities with considerable abundances, indicating that they may be responsible for ACE biodegradation in the enrichments. Besides, two ACE-degrading pure strains, affiliated to the genus Chelatococcus, were isolated from the enrichment. The genomic analysis showed that these two isolates were the new species that were genetically distinct from their relatives. Two type strains, Chelatococcus asaccharovorans DSM 6462 and Chelatococcus composti DSM 101465, could not degrade ACE, implying that the ACE-degrading capability was not shared among the different species in the genus Chelatococcus. The results of the degradation experiment showed that the two isolates could use ACE as the sole carbon source and mineralize ~90% of the total organic carbon. Three biotransformation products (TP96, TP180B, and TP182B) were demonstrated by UPLC-QTOF-MS. The results of this study provide valuable insights into ACE biodegradation and its biotransformation products.

Organic contaminants of emerging concern in leachate of historic municipal landfills.

Many types of contaminants of emerging concern (CECs), including per- and poly-fluoroalkyl substances (PFAS), have been found in leachate of operating municipal landfills. However, there is only limited information on CECs presence in leachate of historic landfills (≥3 decades since closure, often lacking engineered liners or leachate collection systems) at concentrations that may pose a risk to nearby wells and surface water ecosystems. In this study, 48 samples of leachate-impacted groundwater were collected from 20 historic landfills in Ontario, Canada. The CECs measured included artificial sweeteners (ASs), PFAS, organophosphate esters (OPE), pharmaceuticals, bisphenols, sulfamic acid, perchlorate, and substituted phenols. The common presence of the AS saccharin, a known indicator of old landfill leachate, combined with mostly negligible levels of the AS acesulfame, an indicator of modern wastewater, revealed that most samples were strongly influenced by leachate and not cross-contaminated by wastewater (which can contain these same CECs). Several landfills, including ones closed in the 1960s, had total PFAS concentrations similar to those previously measured at modern landfills, with a maximum observed here of 12.7μg/L. Notably elevated concentrations of several OPE, sulfamic acid, cotinine, and bisphenols A and S were found at many 30-60 year-old landfills. There was little indication of declining concentrations with landfill age, suggesting historic landfills can be long-term sources of CECs to groundwater and that certain CECs may be useful tracers for historic landfill leachate. These findings provide guidance on which CECs may require monitoring at historic landfill sites and wastewater treatment plants receiving their effluent.