Sulfamethoxazole Enhances Specific Enzymatic Activities under Aerobic Heterotrophic Conditions: A Metaproteomic Approach.

The growing concern about antibiotic-resistant microorganisms has focused on the sludge from wastewater treatment plants (WWTPs) as a potential hotspot for their development and spread. To this end, it seems relevant to analyze the changes on the microbiota as a consequence of the antibiotics that wastewater may contain. This study aims at determining whether the presence of sulfamethoxazole (SMX), even in relatively low concentrations, modifies the microbial activities and the enzymatic expression of an activated sludge under aerobic heterotrophic conditions. For that purpose, we applied a metaproteomic approach in combination with genomic and transformation product analyses. SMX was biotransformed, and the metabolite 2,4(1H,3H)-pteridinedione-SMX (PtO-SMX) from the pterin-conjugation pathway was detected at all concentrations tested. Metaproteomics showed that SMX at 50-2000 µg/L slightly affected the microbial community structure, which was confirmed by DNA metabarcoding. Interestingly, an enhanced activity of the genus Corynebacterium and specifically of five enzymes involved in its central carbon metabolism was found at increased SMX concentrations. Our results suggest a role of Corynebacterium genus on SMX risks mitigation in our bioreactors.

Enzymatic cometabolic biotransformation of organic micropollutants in wastewater treatment plants: A review.

Biotransformation of trace-level organic micropollutants (OMPs) by complex microbial communities in wastewater treatment facilities is a key process for their detoxification and environmental impact reduction. Therefore, understanding the metabolic activities and mechanisms that contribute to their biotransformation is essential when developing approaches aiming to minimize their discharge. This review addresses the relevance of cometabolic processes and discusses the main enzymatic activities currently known to take part in OMPs removal under different redox environments in the compartments of wastewater treatment plants. Furthermore, the most common methodologies to decipher such enzymes are discussed, including the use of in vitro enzyme assays, enzymatic inhibitors, the analysis of transformation products and the application of several -omic techniques. Finally, perspectives on major challenges and future research requirements to improve OMPs biotransformation are proposed.

Heterotrophic enzymatic biotransformations of organic micropollutants in activated sludge.

While heterotrophic microorganisms constitute the major fraction of activated sludge biomass, the role of heterotrophs in the biotransformation of organic micropollutants (OMPs) has not been fully elucidated. Yet, such knowledge is essential, particularly when conceiving novel wastewater treatment plants based on a two-stage process including an A-stage under heterotrophic conditions and a B-stage based on anammox activity. Biotransformation of OMPs in activated sludge is thought to mostly occur cometabolically thanks to the action of low specificity enzymes involved in the metabolism of the primary substrates. For a better understanding of the process, it is important to determine such enzymatic activities and the underlying mechanisms involved in OMPs biotransformation. This task has proven to be difficult due to the lack of information about the enzymatic processes and the complexity of the biological systems present in activated sludge. In this paper, a continuous aerobic heterotrophic reactor following 20 OMPs at environmental concentrations was operated to (i) assess the potential of heterotrophs during the cometabolic biotransformation of OMPs, (ii) identify biotransformation reactions catalyzed by aerobic heterotrophs and (iii) predict possible heterotrophic enzymatic activities responsible for such biotransformations. Contradicting previous reports on the dominant role of nitrifiers in OMPs removal during activated sludge treatment, the heterotrophic population proved its capacity to biotransform the OMPs to extents equivalent to reported values in nitrifying activated sludge plants. Besides, 12 transformation products potentially formed through the activity of several enzymes present in heterotrophs, including monooxygenases, dioxygenases, hydrolases and transferases, were identified.

The organic loading rate affects organic micropollutants' cometabolic biotransformation kinetics under heterotrophic conditions in activated sludge.

Several studies have shown that organic micropollutants (OMPs) are biotransformed cometabolically in activated sludge systems. However, the individual role of heterotrophs in the microbial consortium is still not clear, i.e., there is still a gap regarding the influence of the heterotrophic activity on the cometabolic biotransformation kinetics and yield of the OMPs. Aiming to answer these questions, experiments with increasing primary substrate concentrations were performed under aerobic heterotrophic conditions in a continuous stirred tank reactor operated at several organic loading rates (OLR) with fixed hydraulic retention time. Moreover, the individual kinetic parameters were determined in batch assays with different initial substrate concentrations using the sludges from the continuous reactor. A set of 15 OMPs displaying a variety of physicochemical properties were spiked to the feeding in the ng L-1 - µg L-1 range. Results reveal that the biodegradation of the primary carbon source and the biotransformation of the OMPs occur simultaneously, in clear evidence of cometabolic behavior. Moreover, we conclude that the OMPs biotransformation kinetic constant (kbiol) shows a linear dependence with the OLR of the primary substrate for most of the compounds studied, suggesting that the heterotrophic activity seriously affects the OMPs biotransformation kinetics. However, under typical activated sludge systems operating conditions (hydraulic retention times above 8 h), their biotransformation yield would not be significantly affected.