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-ß-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.

Micropollutant transformation and taxonomic composition in hybrid MBBR - A comparison of carrier-attached biofilm and suspended sludge.

The suspended sludge and carrier-attached biofilms of three different hybrid moving bed biofilm reactor (MBBR) systems were investigated with respect to their transformation potential for a broad range of micropollutants (MPs) as well as their microbial community composition. For this purpose, laboratory-scale batch experiments were conducted with the separated suspended sludge and the carrier-attached biofilm of every system in triplicate. For all batches the removal of 31 MPs as well as the composition of the microbial community were analyzed. The carrier-attached biofilms from two hybrid MBBR systems showed a significant higher overall transformation potential in comparison to the respective suspended sludge. Especially for the MPs trimethoprim, diclofenac, mecoprop, climbazole and the human metabolite 10,11-dihydro-10-hydroxycarbamazepine consistently higher pseudo-first-order transformation rates could be observed in all three systems. The analysis of the taxonomic composition revealed taxa showing higher relative abundances in the carrier-attached biofilms (e. g. Nitrospirae and Chloroflexi) and in the suspended biomasses (e. g. Bacteroidetes and Betaproteobacteria). Correlations of the biodiversity indices and the MP biotransformation rates resulted in significant positive associations for 11 compounds in suspended sludge, but mostly negative associations for the carrier-attached biofilms. The distinct differences in MP removal between suspended sludge and carrier-attached biofilm of the three different MBBR systems were also reflected by a statistically significant link between the occurrence of specific bacterial taxa (Acidibacter, Nitrospira and Rhizomicrobium) and MP transformation rates of certain MPs. Even though the identified correlations might not necessarily be of causal nature, some of the identified taxa might serve as suitable indicators for the transformation potential of suspended sludge or carrier-attached biofilms.

Biological transformation of fexofenadine and sitagliptin by carrier-attached biomass and suspended sludge from a hybrid moving bed biofilm reactor.

Laboratory-scale experiments were conducted to investigate the (bio)transformation of the antidiabetic sitagliptin (STG) and the antihistamine fexofenadine (FXF) during wastewater treatment. As inoculum either attached-growth on carriers or suspended sludge from a hybrid moving bed biofilm reactor (HMBBR) was used. Both target compounds were incubated in degradation experiments and quantified via LC-MS/MS for degradation kinetics. Furthermore transformation products (TPs) were analyzed via high resolution mass spectrometry (HRMS). Structural elucidation of the TPs was based on the high resolution molecular ion mass to propose a molecular formula and on MS2 fragmentation to elucidate the chemical structure of the TPs. In total, 22 TPs (9 TPs for STG and 13 TPs for FXF) were detected in the experiments with STG and FXF. For all TPs, chemical structures could be proposed. STG was mainly transformed via amide hydrolysis and conjugation of the primary amine moiety. In contrast, FXF was predominantly transformed by oxidative reactions such as oxidation (dehydrogenation) and hydroxylation. Furthermore, FXF was removed significantly faster in contact with carriers compared to suspended sludge, whereas STG was degraded slightly faster in contact with suspended sludge. Moreover, the primary TP of FXF was also degraded faster in contact with carriers leading to higher proportions of secondary TPs. Thus, the microbial community of both carriers and suspended sludge catalyzed the same primary transformation reactions but the transformation kinetics of FXF and the formation/degradation of FXF TPs were considerably higher in contact with carrier-attached biomass. The primary degradation of both target compounds in pilot- and full-scale conventional activated sludge (CAS) and MBBR reactors reached 42 and 61% for FXF and STG, respectively. Up to three of the identified TPs of FXF and 8 TPs of STG were detected in the effluents of pilot- and full-scale CAS and MBBR.

Extended anaerobic conditions in the biological wastewater treatment: Higher reduction of toxicity compared to target organic micropollutants.

Extended anaerobic conditions during biological wastewater treatment may enhance the biodegradation of micropollutants. To explore this, we combined iron-reducing or substrate-limited anaerobic conditions and aerobic pilot-scale reactors directly at a wastewater treatment plant. To investigate the detoxification by these processes, we applied two in vitro bioassays for baseline toxicity (Microtox) and reactive toxicity (AREc32) as well as in vivo bioassays with aquatic model species in two laboratory experiments (Desmodesmus subspicatus, Daphnia magna) and two on-site, flow-through experiments (Potamopyrgus antipodarum, Lumbriculus variegatus). Moreover, we analyzed 31 commonly occurring micropollutants and 10 metabolites. The baseline toxicity of raw wastewater was effectively removed in full-scale and reactor scale activated sludge treatment (>85%), while the oxidative stress response was only partially removed (>61%). A combination of an anaerobic pre-treatment under iron reducing conditions and an aerobic nitrification significantly further reduced the residual in vitro toxicities by 46-60% and outperformed the second combination consisting of an aerobic pre-treatment and an anaerobic post-treatment under substrate-limiting conditions (27-43%). Exposure to effluents of the activated sludge treatment did not induce adverse in vivo effects in aquatic invertebrates. Accordingly, no further improvement in water quality could be observed. Compared to that, the removal of persistent micropollutants was increased. However, this observation was restricted to a limited number of compounds and the removal of the sum concentration of all target micropollutants was relative low (14-17%). In conclusion, combinations of strictly anaerobic and aerobic processes significantly enhanced the removal of specific and non-specific in vitro toxicities. Thus, an optimization of biological wastewater treatment can lead to a substantially improved detoxification. These otherwise hidden capacities of a treatment technology can only be uncovered by a complementary biological analysis.

Biodegradation of the artificial sweetener acesulfame in biological wastewater treatment and sandfilters.

A considerable removal of the artificial sweetener acesulfame (ACE) was observed during activated sludge processes at 13 wastewater treatment plants (WWTPs) as well as in a full-scale sand filter of a water works. A long-term sampling campaign over a period of almost two years revealed that ACE removal in WWTPs can be highly variable over time. Nitrifying/denitrifying sequencing batch reactors (SBR) as well as aerobic batch experiments with activated sludge and filter sand from a water works confirmed that both activated sludge as well as filter sand can efficiently remove ACE and that the removal can be attributed to biologically mediated degradation processes. The lab results strongly indicated that varying ACE removal in WWTPs is not associated with nitrification processes. Neither an enhancement of the nitrification rate nor the availability of ammonium or the inhibition of ammonium monooxygenase by N-allylthiourea (ATU) affected the degradation. Moreover, ACE was found to be also degradable by activated sludge under denitrifying conditions, while being persistent in the absence of both dissolved oxygen and nitrate. Using ion chromatography coupled with high resolution mass spectrometry, sulfamic acid (SA) was identified as the predominant transformation product (TP). Quantitative analysis of ACE and SA revealed a closed mass balance during the entire test period and confirmed that ACE was quantitatively transformed to SA. Measurements of dissolved organic carbon (DOC) revealed an almost complete removal of the carbon originating from ACE, thereby further confirming that SA is the only relevant final TP in the assumed degradation pathway of ACE. A first analysis of SA in three municipal WWTP revealed similar concentrations in influents and effluents with maximum concentrations of up to 2.3 mg/L. The high concentrations of SA in wastewater are in accordance with the extensive use of SA in acid cleaners, while the degradation of ACE in WWTPs adds only a very small portion of the total load of SA discharged into surface waters. No removal of SA was observed by the biological treatment applied at these WWTPs. Moreover, SA was also stable in the aerobic batch experiments conducted with the filter sand from a water works. Hence, SA might be a more appropriate wastewater tracer than ACE due to its chemical and microbiological persistence, the negligible sorbing affinity (high negative charge density) and its elevated concentrations in WWTP effluents.

Tracing the limits of organic micropollutant removal in biological wastewater treatment.

Removal of organic micropollutants was investigated in 15 diverse biological reactors through short and long-term experiments. Short-term batch experiments were performed with activated sludge from three parallel sequencing batch reactors (25, 40, and 80 d solid retention time, SRT) fed with synthetic wastewater without micropollutants for one year. Despite the minimal micropollutant exposure, the synthetic wastewater sludges were able to degrade several micropollutants present in municipal wastewater. The degradation occurred immediately after spiking (1-5 µg/L), showed no strong or systematic correlation to the sludge age, and proceeded at rates comparable to those of municipal wastewater sludges. Thus, the results from the batch experiments indicate that degradation of organic micropollutants in biological wastewater treatment is quite insensitive to SRT increases from 25 to 80 days, and not necessarily induced by exposure to micropollutants. Long-term experiments with municipal wastewater were performed to assess the potential for extended biological micropollutant removal under different redox conditions and substrate concentrations (carbon and nitrogen). A total of 31 organic micropollutants were monitored through influent-effluent sampling of twelve municipal wastewater reactors. In accordance with the results from the sludges grown on synthetic wastewater, several compounds such as bezafibrate, atenolol and acyclovir were significantly removed in the activated sludge processes fed with municipal wastewater. Complementary removal of two compounds, diuron and diclofenac, was achieved in an oxic biofilm treatment. A few aerobically persistent micropollutants such as venlafaxine, diatrizoate and tramadol were removed under anaerobic conditions, but a large number of micropollutants persisted in all biological treatments. Collectively, these results indicate that certain improvements in biological micropollutant removal can be achieved by combining different aerobic and anaerobic treatments, but that these improvements are restricted to a limited number of compounds.

Advancing Biological Wastewater Treatment: Extended Anaerobic Conditions Enhance the Removal of Endocrine and Dioxin-like Activities.

Conventional activated sludge treatment of wastewater does not completely remove micropollutants. Here, extending anaerobic conditions may enhance biodegradation. To explore this, we combined iron-reducing or substrate-limiting and aerobic pilot-scale reactors directly at a wastewater treatment plant. To assess the removal of endocrine disrupting chemicals (EDCs) as group of micropollutants that adversely affects wildlife, we applied a bioanalytical approach. We used in vitro bioassays covering seven receptor-mediated mechanisms of action, including (anti)androgenicity, (anti)estrogenicity, retinoid-like, and dioxin-like activity. Untreated wastewater induced antiandrogenic, estrogenic, antiestrogenic, and retinoid-like activity. Full-scale as well as reactor-scale activated sludge treatment effectively removes the observed effects. Nevertheless, high antiandrogenic and minor dioxin-like and estrogenic effects persisted in the treated effluent that may still be environmentally relevant. The anaerobic post-treatment under substrate-limiting conditions resulted in an additional removal of endocrine activities by 17-40%. The anaerobic pre-treatment under iron-reducing conditions significantly enhanced the removal of the residual effects by 40-75%. In conclusion, this study demonstrates that a further optimization of biological wastewater treatment is possible. Here, implementing iron-reducing anaerobic conditions preceding aerobic treatment appears promising to improve the removal of receptor-mediated toxicity.

New insights into the transformation of trimethoprim during biological wastewater treatment.

The antibiotic trimethoprim (TMP), a micropollutant found at µg/L levels in raw wastewater, was investigated with regard to its (bio)transformation during biological wastewater treatment. A pilot-scale, nitrifying/denitrifying Sequencing Batch Reactor (SBR) fed with municipal wastewater was monitored for TMP removal during a 16-month monitoring study. Laboratory-scaled bioreactors spiked with TMP were applied to identify the transformation products (TPs). In total, six TPs could be identified from TMP. However, the TP formation was influenced by the spike concentration. At an initial concentration of 500 µg/L TMP, only two TPs were found, whereas at 5 µg/L a completely different transformation pathway led to four further TPs. At low concentrations, TMP was demethylated forming 4-desmethyl-TMP, which was then quickly hydroxylated, oxidized and cleaved forming 2,4-diaminopyrimidine-5-carboxylic acid (DAPC) via two intermediate TPs. DAPC was detected in the SBR effluent in a 3-d composite sample with 61 ng/L, which accounts for 52% of the attenuated TMP. The primary degradation at low spiking levels was best modelled by a pseudo-first order kinetic. Considering the SBR, the model predicted a TMP removal of 88-94% for the reactor, consistent with a monitoring campaign exhibiting an average removal of >83%. Both the TP formation profiles and kinetic modelling indicated that only the results from the bioreactor tests at low spike concentrations were representative of the transformation in the SBR.