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There is increasing public concern regarding the fate of antibiotic resistance genes (ARGs) during wastewater treatment, their persistence during the treatment process and their potential impacts on the receiving water bodies. In this study, we used quantitative PCR (qPCR) to determine the abundance of nine ARGs and a class 1 integron associated integrase gene in 16 wastewater treatment plant (WWTP) effluents from ten different European countries. In order to assess the impact on the receiving water bodies, gene abundances in the latter were also analysed. Six out of the nine ARGs analysed were detected in all effluent and river water samples. Among the quantified genes, intI1 and sul1 were the most abundant. Our results demonstrate that European WWTP contribute to the enrichment of the resistome in the receiving water bodies with the particular impact being dependent on the effluent load and local hydrological conditions. The ARGs concentrations in WWTP effluents were found to be inversely correlated to the number of implemented biological treatment steps, indicating a possible option for WWTP management. Furthermore, this study has identified bla OXA-58 as a possible resistance gene for future studies investigating the impact of WWTPs on their receiving water., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Antibiotics-bacteria interactions depend on antibiotic concentration at the scale of bacteria. This study investigates how vancomycin penetrates into activated sludge flocs and can be sorbed on the bacteria and extracellular polymeric substances (EPS). The 3D structure of flocs was imaged using EPS autofluorescence. The green fluorescent BODIPY ® FL vancomycin was introduced in a microscopic chamber containing activated sludge and penetration of vancomycin into the flocs by diffusion was observed using time-lapse microscopy. The penetration depended on the floc structure, as long and large pores could go through the whole flocs making preferential path. The antibiotic concentration into the flocs was also found to depend on the sorption rate. BODIPY ® FL vancomycin was found to bind preferentially into Gram + bacteria than on EPS. The vancomycin adsorption constant on bacteria according to the linear adsorption model, Kd bacteria was estimated to be 5 times higher (SD 2.6) than the adsorption constant on EPS Kd EPS . These results suggest that antibiotic removal by sorption into wastewater treatment plants could change according to the amount of bacteria in the sludge. Moreover, antibiotic concentration at the scale of bacteria could be significantly higher than the concentration in the bulk solution and this should be taken into account when studying antibiotic activity or biodegradation., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

This study investigated the chronic impact of sulfamethoxazole (SMX) on activated sludge sustaining an enriched nitrifying biomass. For this purpose, a laboratory scale fill and draw reactor was operated with 100 mg COD/L of peptone mixture and 50 mg N/L of ammonia at a sludge age of 15 days. Additionally, the biomass was exposed to a daily SMX dose of 50 mg/L once the reactor reached steady-state conditions. The reactor performance and microbial composition were monitored for 37 days with conventional parameters and molecular techniques based on the gene for ammonia monooxygenase subunit A (amoA) and the prokaryotic 16S rRNA gene. Denaturing gradient gel electrophoresis (DGGE) and 16S rRNA gene cloning analyses suggested a microbial community change concurrent with the addition of SMX. Specifically, quantitative polymerase chain reaction analyses (qPCR/RT-qPCR) revealed a significant reduction in the levels and activity of ammonia oxidizing bacteria (AOB). However, the acclimation period ended with high amoA mRNA levels and improved nitrification efficiency. Partial degradation of SMX by heterotrophic bacteria was also observed., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

This study evaluated the chronic impact of tetracycline on biomass with enriched nitrifying community sustained in a lab-scale activated sludge system. For this purpose, a fill and draw reactor fed with 100 mg COD/L of peptone mixture and 50 mg N/L of ammonia was sustained at a sludge age of 15 days. At steady-state, the reactor operation was continued with a daily tetracycline dosing of 50 mg/L for more than 40 days, with periodic monitoring of the microbial composition, the nitrifying bacteria abundance, as well as the amoA and 16S rRNA gene activity, using molecular techniques. Changes in the kinetics of nitrification were quantified by modelling concentration profiles of major nitrogen fractions and oxygen uptake rate profiles derived from parallel batch experiments. Activated sludge modeling results indicated inhibitory impact of tetracycline on the growth of nitrifiers with a significant increase of the half saturation coefficients in corresponding rate equations. Tetracycline also inactivated biomass components of the enriched culture at a gradually increasing rate with time of exposure, leading to total collapse of nitrification. Molecular analyses revealed significant changes in the composition of the microbial community throughout the observation period. They also showed that continuous exposure to tetracycline inflicted significant reduction in amoA mRNA and 16S rRNA levels directly affecting nitrification. The chronic impact was much more pronounced on the ammonia oxidizing bacteria (AOB) community. These observations explained the basis of numerical changes identified in the growth kinetics of nitrifiers under stress conditions., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

In many biological wastewater treatment systems, bacterial growth and the amount of active biomass are limited by the availability of substrate. Under these low growth conditions, endogenous processes have a significant influence on the amount of active biomass and therefore, the overall system performance. In enhanced biological phosphorus removal (EBPR) systems endogenous processes can also influence the levels of the internal storage compounds of the polyphosphate accumulating organisms (PAO), directly affecting phosphorus removal performance. The purpose of this study was to evaluate the significance of different endogenous processes that occur during the long-term starvation of EBPR sludge under aerobic and anaerobic conditions. Activated sludge obtained from a laboratory sequencing batch reactor was used to perform a series of batch starvation experiments. Under aerobic starvation conditions we observed a significant decay of PAO (first-order decay rate of 0.15/d) together with a rapid utilization of polyhydroxyalkanoates (PHA) and a slower utilization of glycogen and polyphosphate to generate maintenance energy. On the other hand, anaerobic starvation was best described by maintenance processes that rapidly reduce the levels of polyphosphate and glycogen under starvation conditions while no significant decay of PAO was observed. The endogenous utilization of glycogen for maintenance purposes is currently not included in available EBPR models. Our experimental results suggest that mathematical models for in EBPR should differentiate between aerobic and anaerobic endogenous processes, as they influence active biomass and storage products differently.

Residence time distribution experiments have been performed on an activated sludge 3000 m3 channel reactor aerated by gas diffusion (for different liquid flowrates under constant aeration rate and constant water depth) and on a bench-scale channel reactor aerated from the bottom (for different liquid and gas flowrates and water depths) in order to characterize their hydrodynamics. Both units can be modeled as plug flow reactors with axial dispersion. A general correlation has been obtained to predict the axial dispersion coefficient as a function of the gas and liquid velocities and the geometrical parameters of the full-scale and bench-scale reactors. Finally, to facilitate the simulation of biological reactions in transient state, an equivalent model based on tanks-in-series with variable back-mixing flowrate is proposed.

Microscopic techniques ranging from epifluorescence microscopy to confocal laser scanning microscopy (CLSM) and two photon excitation laser scanning microscopy (TPE-LSM) combined with fluorescent stains can help to evaluate complex microbial aggregates such as activated sludge flocs. To determine the application limits of these microscopic techniques, activated sludge samples from three different sources were evaluated after staining with a fluorescent viability indicator (Baclight Bacterial Viability Kit, Molecular Probes). Image analysis routines were developed to quantify overall amounts of red and green stained cells, location of stained cells within the flocs, and the spatial organization in clusters and filaments. It was found that the selection of the appropriate microscopic technique depends strongly on the type of microbial aggregates being analyzed. For flocs with high cell density, the use of TPE-LSM is preferred, since it provides a clearer image of the internal structure of the aggregate. Epifluorescence microscopy did not allow to reliably quantify red stained cells in dense aggregates. CLSM did not adequately image the internal filamentous structure and the location of stained cells within dense flocs. However, for typical activated sludge flocs epifluorescence and CLSM proved adequate.