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Recently, concerns have been raised that residues of glyphosate-based herbicides may interfere with the homeostasis of the intestinal bacterial community and thereby affect the health of humans or animals. The biochemical pathway for aromatic amino acid synthesis (Shikimate pathway), which is specifically inhibited by glyphosate, is shared by plants and numerous bacterial species. Several in vitro studies have shown that various groups of intestinal bacteria may be differently affected by glyphosate. Here, we present results from an animal exposure trial combining deep 16S rRNA gene sequencing of the bacterial community with liquid chromatography mass spectrometry (LC-MS) based metabolic profiling of aromatic amino acids and their downstream metabolites. We found that glyphosate as well as the commercial formulation Glyfonova450 PLUS administered at up to fifty times the established European Acceptable Daily Intake (ADI = 0.5 mg/kg body weight) had very limited effects on bacterial community composition in Sprague Dawley rats during a two-week exposure trial. The effect of glyphosate on prototrophic bacterial growth was highly dependent on the availability of aromatic amino acids, suggesting that the observed limited effect on bacterial composition was due to the presence of sufficient amounts of aromatic amino acids in the intestinal environment. A strong correlation was observed between intestinal concentrations of glyphosate and intestinal pH, which may partly be explained by an observed reduction in acetic acid produced by the gut bacteria. We conclude that sufficient intestinal levels of aromatic amino acids provided by the diet alleviates the need for bacterial synthesis of aromatic amino acids and thus prevents an antimicrobial effect of glyphosate in vivo. It is however possible that the situation is different in cases of human malnutrition or in production animals.

Addition of external carbon sources to post-denitrification systems is frequently used in wastewater treatment plants to enhance nitrate removal. However, little is known about the fate of micropollutants in post-denitrification systems and the influence of external carbon dosing on their removal. In this study, we assessed the effects of two different types and availability of commonly used carbon sources -methanol and ethanol- on the removal of micropollutants in biofilm systems. Two laboratory-scale moving bed biofilm reactors (MBBRs), containing AnoxKaldnes K1 carriers with acclimated biofilm from full-scale systems, were operated in continuous-flow using wastewater dosed with methanol and ethanol, respectively. Batch experiments with 22 spiked pharmaceuticals were performed to assess removal kinetics. Acetyl-sulfadiazine, atenolol, citalopram, propranolol and trimethoprim were easily biotransformed in both MBBRs (biotransformations rate constants k between 1.2 and 12.9 L g d), 13 compounds were moderately biotransformed (rate constants between 0.2 and 2 L g d) and 4 compounds were recalcitrant. The methanol-dosed MBBR showed higher k (e.g., 1.5-2.5-fold) than in the ethanol-dosed MBBR for 9 out of the 22 studied compounds, equal k for 10 compounds, while 3 compounds (i.e., targeted sulfonamides) were biotransformed faster in the ethanol-dosed MBBR. While biotransformation of most of the targeted compounds followed first-order kinetics, removal of venlafaxine, carbamazepine, sulfamethoxazole and sulfamethizole could be described with a cometabolic model. Analyses of the microbial composition in the biofilms using 16S rRNA amplicon sequencing revealed that the methanol-dosed MBBR contained higher microbial richness than the one dosed with ethanol, suggesting that improved biotransformation of targeted compounds could be associated with higher microbial richness. During continuous-flow operation, at conditions representative of full-scale denitrification systems (hydraulic residence time = 2 h), the removal efficiencies of micropollutants were below 35% in both MBBRs, with the exception of atenolol and trimethoprim (>80%). Overall, this study demonstrated that MBBRs used for post-denitrification could be optimized to enhance the biotransformation of a number of micropollutants by accounting for optimal carbon sources and extended residence time.

Highlights:  Ozone dosage was determined for pharmaceuticals removal in hospital wastewater  The ozone dosage required varied 2-fold with both DOC and pH experienced over time  DOC normalized ozone dosage for 90% removal of 32 pharmaceuticals was determined  At low pH, pharmaceuticals need less ozone while ozone lifetime increased to 20 min  H 2 O 2 dosing shorten the ozone lifetime at low pH to 5 min similar to neutral pH 2 Abstract: Ozonation aimed at removing pharmaceuticals was studied in an effluent from an experimental pilot system using staged Moving Bed Biofilm Reactor (MBBR) tanks for the optimal biological treatment of wastewater from a medical care unit of Aarhus University Hospital. Dissolved Organic Carbon (DOC) and pH in samples varied considerably, and the effect of these two parameters on ozone lifetime and the efficiency of ozone in removing pharmaceuticals were determined. The pH in the effluent varied from 5.0 to 9.0 resulting in approximately a doubling of the required ozone dose at the highest pH for each pharmaceutical. DOC varied from 6 to 20 mg-DOC/L. The ozone required for removing each pharmaceutical, varied linearly with DOC and thus, ozone doses normalized to DOC (specific ozone dosis) agreed between water samples (typically within 15%). At neutral pH the specific ozone dose required to remove the easiest degradable pharmaceutical, sulfadiazine, was 0.50±0.04 mg-O 3 /mg-DOC and the most recalcitrant, diatrizoic acid, required 4.7±0.6 mg-O 3 /mg-DOC. The lifetime of ozone increased drastically in the higher end of the indicated dosage. At the lowest observed pH of 5.0, its lifetime was quadrupled to 20 min which influences the design of the reaction tank. The addition of 0.1 mg-H 2 O 2 per 1 mg-O 3 mitigated the prolonged lifetime without a corresponding influence in the pharmaceutical removal efficiency of ozone.

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Due to the limited efficiency of conventional biological treatment, innovative solutions are being explored to improve the removal of trace organic chemicals in wastewater. Controlling biomass exposure to growth substrate represents an appealing option for process optimization, as substrate availability likely impacts microbial activity, hence organic trace chemical removal. This study investigated the elimination of pharmaceuticals in pre-denitrifying moving bed biofilm reactors (MBBRs), where biofilm exposure to different organic substrate loading and composition was controlled by reactor staging. A three-stage MBBR and a single-stage reference MBBR (with the same operating volume and filling ratio) were operated under continuous-flow conditions (18 months). Two sets of batch experiments (day 100 and 471) were performed to quantify and compare pharmaceutical removal and denitrification kinetics in the different MBBRs. Experimental results revealed the possible influence of retransformation (e.g., from conjugated metabolites) and enantioselectivity on the removal of selected pharmaceuticals. In the second set of experiments, specific trends in denitrification and biotransformation kinetics were observed, with highest and lowest rates/rate constants in the first (S1) and the last (S3) staged sub-reactors, respectively. These observations were confirmed by removal efficiency data obtained during continuous-flow operation, with limited removal (<10%) of recalcitrant pharmaceuticals and highest removal in S1 within the three-stage MBBR. Notably, biotransformation rate constants obtained for non-recalcitrant pharmaceuticals correlated with mean specific denitrification rates, maximum specific growth rates and observed growth yield values. Overall, these findings suggest that: (i) the long-term exposure to tiered substrate accessibility in the three-stage configuration shaped the denitrification and biotransformation capacity of biofilms, with significant reduction under substrate limitation; (ii) biotransformation of pharmaceuticals may have occurred as a result of cometabolism by heterotrophic denitrifying bacteria.

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