Sequential Anaerobic-Aerobic Treatment Enhances Sulfamethoxazole Removal: From Batch Cultures to Observations in a Large-Scale Wastewater Treatment Plant.

Sulfamethoxazole (SMX) passes through conventional wastewater treatment plants (WWTPs) mainly unaltered. Under anoxic conditions sulfate-reducing bacteria can transform SMX but the fate of the transformation products (TPs) and their prevalence in WWTPs remain unknown. Here, we report the anaerobic formation and aerobic degradation of SMX TPs. SMX biotransformation was observed in nitrate- and sulfate-reducing enrichment cultures. We identified 10 SMX TPs predominantly showing alterations in the heterocyclic and N4-arylamine moieties. Abiotic oxic incubation of sulfate-reducing culture filtrates led to further degradation of the major anaerobic SMX TPs. Upon reinoculation under oxic conditions, all anaerobically formed TPs, including the secondary TPs, were degraded. In samples collected at different stages of a full-scale municipal WWTP, anaerobically formed SMX TPs were detected at high concentrations in the primary clarifier and digested sludge units, where anoxic conditions were prevalent. Contrarily, their concentrations were lower in oxic zones like the biological treatment and final effluent. Our results suggest that anaerobically formed TPs were eliminated in the aerobic treatment stages, consistent with our observations in batch biotransformation experiments. More generally, our findings highlight the significance of varying redox states determining the fate of SMX and its TPs in engineered environments.

Direct Phototransformation of Sulfamethoxazole Characterized by Four-Dimensional Element Compound Specific Isotope Analysis.

The antibiotic sulfamethoxazole (SMX) undergoes direct phototransformation by sunlight, constituting a notable dissipation process in the environment. SMX exists in both neutral and anionic forms, depending on the pH conditions. To discern the direct photodegradation of SMX at various pH levels and differentiate it from other transformation processes, we conducted phototransformation of SMX under simulated sunlight at pH 7 and 3, employing both transformation product (TP) and compound-specific stable isotope analyses. At pH 7, the primary TPs were sulfanilic acid and 3A5MI, followed by sulfanilamide and (5-methylisoxazol-3-yl)-sulfamate, whereas at pH 3, a photoisomer was the dominant product, followed by sulfanilic acid and 3A5MI. Isotope fractionation patterns revealed normal 13C, 34S, and inverse 15N isotope fractionation, which exhibited significant differences between pH 7 and 3. This indicates a pH-dependent transformation process in SMX direct phototransformation. The hydrogen isotopic composition of SMX remained stable during direct phototransformation at both pH levels. Moreover, there was no variation observed in 33S between the two pH levels, indicating that the 33S mass-independent process remains unaffected by changes in pH. The analysis of main TPs and single-element isotopic fractionation suggests varying combinations of bond cleavages at different pH values, resulting in distinct patterns of isotopic fractionation. Conversely, dual-element isotope values at different pH levels did not significantly differ, indicating cleavage of several bonds in parallel. Hence, prudent interpretation of dual-element isotope analysis in these systems is warranted. These findings highlight the potential of multielement compound-specific isotope analysis in characterizing pH-dependent direct phototransformation of SMX, thereby facilitating the evaluation of its natural attenuation through sunlight photolysis in the environment.

Recently evolved combination of unique sulfatase and amidase genes enables bacterial degradation of the wastewater micropollutant acesulfame worldwide.

Xenobiotics often challenge the principle of microbial infallibility. One example is acesulfame introduced in the 1980s as zero-calorie sweetener, which was recalcitrant in wastewater treatment plants until the early 2010s. Then, efficient removal has been reported with increasing frequency. By studying acesulfame metabolism in alphaproteobacterial degraders of the genera Bosea and Chelatococcus, we experimentally confirmed the previously postulated route of two subsequent hydrolysis steps via acetoacetamide-N-sulfonate (ANSA) to acetoacetate and sulfamate. Genome comparison of wildtype Bosea sp. 100-5 and an acesulfame degradation-defective mutant revealed the involvement of two plasmid-borne gene clusters. The acesulfame-hydrolyzing sulfatase is strictly manganese-dependent and belongs to the metallo beta-lactamase family. In all degraders analyzed, it is encoded on a highly conserved gene cluster embedded in a composite transposon. The ANSA amidase, on the other hand, is an amidase signature domain enzyme encoded in another gene cluster showing variable length among degrading strains. Transposition of the sulfatase gene cluster between chromosome and plasmid explains how the two catabolic gene clusters recently combined for the degradation of acesulfame. Searching available genomes and metagenomes for the two hydrolases and associated genes indicates that the acesulfame plasmid evolved and spread worldwide in short time. While the sulfatase is unprecedented and unique for acesulfame degraders, the amidase occurs in different genetic environments and likely evolved for the degradation of other substrates. Evolution of the acesulfame degradation pathway might have been supported by the presence of structurally related natural and anthropogenic compounds, such as aminoacyl sulfamate ribonucleotide or sulfonamide antibiotics.

Biotransformation of Acetaminophen by intact cells and crude enzymes of bacteria: A comparative study and modelling.

Acetaminophen (APAP), which is an active ingredient of many analgesic drugs, is one of the contaminants of emerging concern in the environment. Although APAP is biodegradable, it is frequently detected in treatment plant effluents, surface water and soil suggesting that there are factors affecting the fate of APAP in the environment. In this study, four strains of bacteria that can degrade APAP were isolated from soil. Those strains belonged to Rhodococcus, Pseudomonas, Flavobacterium, and Sphingobium genera of Bacteria. A series of kinetic experiments were performed on the isolates in shake-flasks to determine biodegradation rate constant as well as the effect of temperature, APAP concentration and cell density on the biodegradation rates. APAP biodegradation follows the first order reaction kinetics which is coupled with cell growth. The specific APAP biodegradation rate constant (k) for all strains was similar and equal to 0.19 ±â€¯0.01 h-1. The temperature, at which APAP biodegradation rate was maximum, was 35 °C. APAP biodegradation rate was linearly correlated with both the initial APAP concentration and the cell density. Initial step of the APAP biodegradation was hydrolysis of the amide bond which resulted in formation and accumulation of p-aminophenol suggesting that aryl acylamidase enzyme is responsible for the biotransformation. In addition, free and immobilized crude enzymes of the isolates transformed APAP at similar rates, comparable to the intact cells. This study showed that APAP biodegradation is achieved by a diverse group of bacteria having a similar enzyme operating at a constant kinetics which is very slow at environmentally relevant APAP concentrations. Natural removal of APAP in the environment is limited by kinetics, therefore APAP-bearing waste streams should be treated in adsorption enhanced biological systems before discharged into the environment.