New Perspectives on the Interactions between Adsorption and Degradation of Organic Micropollutants in Granular Activated Carbon Filters.

The removal of organic micropollutants in granular activated carbon (GAC) filters can be attributed to adsorption and biological degradation. These two processes can interact with each other or proceed independently. To illustrate the differences in their interaction, three 14C-labeled organic micropollutants with varying potentials for adsorption and biodegradation were selected to study their adsorption and biodegradation in columns with adsorbing (GAC) and non-adsorbing (sand) filter media. Using 14CO2 formation as a marker for biodegradation, we demonstrated that the biodegradation of poorly adsorbing N-nitrosodimethylamine (NDMA) was more sensitive to changes in the empty bed contact time (EBCT) compared with that of moderately adsorbing diclofenac. Further, diclofenac that had adsorbed under anoxic conditions could be degraded when molecular oxygen became available, and substantial biodegradation (≥60%) of diclofenac could be achieved with a 15 min EBCT in the GAC filter. These findings suggest that the retention of micropollutants in GAC filters, by prolonging the micropollutant residence time through adsorption, can enable longer time periods for degradations than what the hydraulic retention time would allow for. For the biologically recalcitrant compound carbamazepine, differences in breakthrough between the 14C-labeled and nonradiolabeled compounds revealed a substantial retention via successive adsorption-desorption, which could pose a potential challenge in the interpretation of GAC filter performance.

Biological degradation of organic micropollutants in GAC filters-temporal development and spatial variations.

The capacity for organic micropollutant removal in granular activated carbon (GAC) filters for wastewater treatment changes over time. These changes are in general attributed to changes in adsorption, but may in some cases also be affected by biological degradation. Knowledge on the degradation of organic micropollutants, however, is scarce. In this work, the degradation of micropollutants in several full-scale GAC and sand filters was investigated through incubation experiments over a period of three years, using 14C-labeled organic micropollutants with different susceptibilities to biological degradation (ibuprofen, diclofenac, and carbamazepine), with parallel 16S rRNA gene sequencing. The results showed that the degradation of diclofenac and ibuprofen in GAC filters increased with increasing numbers of bed volumes when free oxygen was available in the filter, while variations over filter depth were limited. Despite relatively large differences in bacterial composition between filters, a degradation of diclofenac was consistently observed for the GAC filters that had been operated with high influent oxygen concentration (DO >8 mg/L). The results of this comprehensive experimental work provide an increased understanding of the interactions between microbial composition, filter material, and oxygen availability in the biological degradation of organic micropollutants in GAC filters.

Effects of conventionally treated and ozonated wastewater on the damselfly larva oxylipidome in response to on-site exposure.

Pharmaceutical residues discharged through insufficiently treated or untreated wastewater enter aquatic environments, where they may adversely impact organisms such as aquatic invertebrates. Ozonation, an advanced wastewater treatment technique, has been successfully implemented to enhance the removal of a broad range of pharmaceuticals, however diverse byproducts and transformation products that are formed during the ozonation process make it difficult to predict how ozonated wastewater may affect aquatic biota. The aim of this study was to investigate effects on fatty acid metabolites, oxylipins, in a common invertebrate species, damselfly larvae, after on-site exposure to conventional wastewater treatment plant (WWTP) effluent and additionally ozonated effluent at a full-scale WWTP. Subsequent ozonation of the conventionally treated wastewater was assessed in terms of i) removal of pharmaceuticals and ii) potential sub-lethal effects on the oxylipidome. Northern damselfly (Coenagrion hastulatum) larvae were exposed for six days in the treatment plant facility to either conventional WWTP effluent or ozonated effluent and the effects on pharmaceutical levels and oxylipin levels were compared with those from tap water control exposure. Ozonation removed pharmaceuticals at an average removal efficiency of 67% (ozone dose of 0.49 g O3/g DOC). Of 38 pharmaceuticals detected in the effluent, 16 were removed to levels below the limit of quantification by ozonation. Levels of two oxylipins, 12(13)-EpODE and 15(16)-EpODE, were reduced in larvae exposed to the conventionally treated wastewater in comparison to the tap water control. 15(16)-EpODE was reduced in the larvae exposed to ozonated effluent in comparison to the tap water control. One oxylipin, 8-HETE, was significantly lower in larvae exposed to conventional WWTP effluent compared to ozonated effluent. In conclusion, the study provides proof-of-principle that damselfly larvae can be used on-site to test the impact of differentially treated wastewater.

Removal of organic micropollutants from municipal wastewater by aerobic granular sludge and conventional activated sludge.

Removal performances of organic micropollutants by conventional activated sludge (CAS) and aerobic granular sludge (AGS) were investigated at a full-scale wastewater treatment plant. Lab-scale kinetic experiments were performed to assess the micropollutant transformation rates under oxic and anoxic conditions. Transformation rates were used to model the micropollutant removal in the full-scale processes. Metagenomic sequencing was used to compare the microbial communities and antimicrobial resistance genes of the CAS and AGS systems. Higher transformation ability was observed for CAS compared to AGS for most compounds, both at the full-scale plant and in the complementary batch experiments. Oxic conditions supported the transformation of several micropollutants with faster and/or comparable rates compared to anoxic conditions. The estimated transformation rates from batch experiments adequately predicted the removal for most micropollutants in the full-scale processes. While the compositions in microbial communities differed between AGS and CAS, the full-scale biological reactors shared similar resistome profiles. Even though granular biomass showed lower potential for micropollutant transformation, AGS systems had somewhat higher gene cluster diversity compared to CAS, which could be related to a higher functional diversity. Micropollutant exposure to biomass or mass transfer limitations, therefore played more important roles in the observed differences in OMP removal.

Microbial bromate reduction following ozonation of bromide-rich wastewater in coastal areas.

Ozonation of wastewater can reduce the release of organic micropollutants, but may result in the formation of undesirable by-products, such as bromate from bromide. Bromide is one of the most abundant ions in seawater, the primary precursor of bromate during ozonation, and the end product in microbial bromate reduction. Investigations were carried out to compare the concentration of bromide in wastewater in coastal and non-coastal catchment areas, to monitor bromate formation during ozonation, and to assess the potential for subsequent bromate reduction with denitrifying carriers. Higher bromide concentrations were systematically observed in wastewater from coastal catchment areas (0.2-2 mg Br-/L) than in wastewater from non-coastal areas (0.06-0.2 mg Br-/L), resulting in elevated formation of bromate during ozonation. Subsequent investigations of bromate reduction in contact with denitrifying carriers from two full-scale moving bed biofilm reactors (MBBRs) showed that 80 % of the bromate formed during ozonation could be reduced to bromide in 60 min with first-order rate constants of 0.3-0.8 L/(gbiomass·h). Flow-through experiments with denitrifying carriers also showed that combined reduction of bromate and nitrate could be achieved below a concentration of 2 mg NOx--N/L. These findings indicate that bromide-rich wastewater is more likely to be of concern when using ozonation in coastal than in non-coastal areas, and that bromate and nitrate reduction can be combined in a single biofilm reactor.

Ozonation of 14C-labeled micropollutants - mineralization of labeled moieties and adsorption of transformation products to activated carbon.

Ozonation transformation products (OTPs) are largely unknown compounds that are formed during the ozonation of micropollutants, and it is uncertain to which extent these compounds can be removed by subsequent adsorption to activated carbon. Thus, 14C-labeled micropollutants were ozonated to generate 14C-labeled OTPs, for which the adsorption of the sum of all 14C-labeled OTPs to activated carbon could be determined, based on the adsorption of the labeled carbon. Further, 14CO2 traps were used to examine the mineralization of 14C-labeled moieties during ozonation. 14CO2-formation revealed a partial mineralization of the 14C-labeled moieties in all compounds except for propyl-labeled bisphenol A and O-methyl-labeled naproxen. A similar degree of mineralization was noted for different compounds labeled at the same moiety, including the carboxylic carbon in diclofenac and ibuprofen (∼40% at 1 g O3/g DOC) and the aniline ring in sulfamethoxazole and sulfadiazine (∼30% at 1 g O3/g DOC). Aromatic ring cleavage was also confirmed for bisphenol A, sulfamethoxazole, and sulfadiazine through the formation of 14CO2. The adsorption experiments demonstrated increased adsorption of micropollutants to powdered activated carbon after ozonation, which was connected to a decreased adsorption of dissolved organic matter (DOM). Conversely, the OTPs showed a substantial and successive decline in adsorption at increased ozone doses for all compounds, likely due to decreased hydrophobicity and aromaticity of the OTPs. These findings indicate that adsorption to activated carbon alone is not a viable removal method for a wide range of ozonation transformation products.

Digging deep into a GAC filter - Temporal and spatial profiling of adsorbed organic micropollutants.

A large pilot-scale granular activated carbon (GAC) filter was operated downstream in a full-scale wastewater treatment plant to remove organic micropollutants. To describe the spatial and temporal developments of micropollutant adsorption profiles in the GAC filter, micropollutants were extracted from GAC media taken at various filter depths and number of treated bed volumes. At a low number of treated bed volumes (2600 BVs), most micropollutants were adsorbed in the top layers of the filter. At increasing number of treated bed volumes (7300-15,500 BVs), the adsorption front for micropollutants progressed through the filter bed at varying rates, with sulfamethoxazole, fluconazole, and PFOS reaching the bottom layer before carbamazepine and other well-adsorbing micropollutants, such as propranolol and citalopram. Higher amounts of adsorbed micropollutants in the bottom layer of the filter bed resulted in decreased removal efficiencies in the treated wastewater. Mass estimations indicated biodegradation for certain micropollutants, such as naproxen, diclofenac, and sulfamethoxazole. A temporary increase in the concentration of the insecticide imidacloprid could be detected in the filter indicating that extraction of adsorbed micropollutants could provide an opportunity for backtracking of loading patterns.

A comparison of adsorption of organic micropollutants onto activated carbon following chemically enhanced primary treatment with microsieving, direct membrane filtration and tertiary treatment of municipal wastewater.

The adsorption of organic micropollutants onto powdered activated carbon (PAC) was investigated in laboratory scale based on samples from four wastewater process streams (matrices); three from a pilot-scale plant with different degrees of physicochemical treatment of municipal wastewater and one from a full-scale activated sludge plant with post-precipitation. The pilot-scale treatment consisted of chemically enhanced primary treatment with microsieving followed by direct membrane filtration as microfiltration or ultrafiltration. The results showed highest adsorption of micropollutants in the tertiary (biologically and chemically) treated wastewater and lowest adsorption in the microsieve filtrate. Adsorption of micropollutants in the direct membrane microfiltration (200 nm) permeate was generally similar to that in the direct membrane ultrafiltration (3 nm) permeate. The higher adsorption of micropollutants in the tertiary treated wastewater could be related to a lower concentration of dissolved organic carbon (DOC) and lower affinity of DOC for PAC at low dosage (<15 mg PAC/L) in this matrix. At a PAC dose of 10 mg/L, sulfamethoxazole was removed by 33% in the tertiary treated wastewater and 7% in the direct membrane microfiltration permeate. In addition to the PAC experiments, a pilot scale sand filter and a proceeding GAC filter was operated on tertiary treated wastewater from the full-scale treatment plant. Similar removal trends in the PAC and GAC experiments were observed when studying a weighted average micropollutant removal in the GAC filter and a similar dose of activated carbon for both PAC and GAC. Positively charged micropollutants were removed to a higher extent than negatively charged ones by both PAC and GAC.

Tracking 14C-Labeled Organic Micropollutants to Differentiate between Adsorption and Degradation in GAC and Biofilm Processes.

Granular activated carbon (GAC) filters can be used to reduce emissions of organic micropollutants via municipal wastewater, but it is still uncertain to which extent biological degradation contributes to their removal in GAC filters. 14C-labeled organic micropollutants were therefore used to distinguish degradation from adsorption in a GAC-filter media with associated biofilm. The rates and extents of biological degradation and adsorption were investigated and compared with other biofilm systems, including a moving bed biofilm reactor (MBBR) and a sand filter, by monitoring 14C activities in the liquid and gas phases. The microbial cleavage of ibuprofen, naproxen, diclofenac, and mecoprop was confirmed for all biofilms, based on the formation of 14CO2, whereas the degradation of 14C-labeled moieties of sulfamethoxazole and carbamazepine was undetected. Higher degradation rates for diclofenac were observed for the GAC-filter media than for the other biofilms. Degradation of previously adsorbed diclofenac onto GAC could be confirmed by the anaerobic adsorption and subsequent aerobic degradation by the GAC-bound biofilm. This study demonstrates the potential use of 14C-labeled micropollutants to study interactions and determine the relative contributions of adsorption and degradation in GAC-based treatment systems.

Integrating dissolved and particulate matter into a prediction tool for ozonation of organic micropollutants in wastewater.

Ozonation is an established technique used to reduce the discharge of organic micropollutants into the aquatic environment, but the possibility of predicting the ozone demand for different wastewater matrices is still limited, especially in the presence of suspended solids (SS). A new tool for the prediction of the removal of organic micropollutants with ozone, based on dissolved and particulate matter in activated sludge effluents, was therefore developed. The removal of 25 organic micropollutants was determined on laboratory scale in the presence and absence of suspended solids. The linear trajectories of the dose-response curves enabled the determination of a new set of removal constants, based on dissolved chemical oxygen demand (COD) and SS. The presence of SS had a more negative effect on the removal of slow-reacting micropollutants (removal constant <3.5 mg CODCr,diss·mg O3-1) with ozone than on the fast-reacting micropollutants (removal constant >3.5 mg CODCr,diss·mg O3-1). However, the decreased removal of the organic micropollutants was generally small, <10%, at typical SS concentrations, <25 mg SS·L-1. Integration of the new removal constants based on COD and SS enabled the removal in an ozone pilot plant to be modelled with an average deviation of <10% for several organic micropollutants. The use of the frequently measured parameters, COD and SS, as input parameters could facilitate the future use of the tool to predict the removal of micropollutants during ozonation.

Promoting the degradation of organic micropollutants in tertiary moving bed biofilm reactors by controlling growth and redox conditions.

A novel process configuration was designed to increase biofilm growth in tertiary moving bed biofilm reactors (MBBRs) by providing additional substrate from primary treated wastewater in a sidestream reactor under different redox conditions in order to improve micropollutant removal in MBBRs with low substrate availability. This novel recirculating MBBR was operated on pilot scale for 13 months, and a systematic increase was seen in the biomass concentration and the micropollutant degradation rates, compared to a tertiary MBBR without additional substrate. The degradation rates per unit carrier surface area increased in the order of ten times, and for certain micropollutants, such as atenolol, metoprolol, trimethoprim and roxithromycin, the degradation rates increased 20-60 times. Aerobic conditions were critical for maintaining high micropollutant degradation rates. With innovative MBBR configurations it may be possible to improve the biological degradation of organic micropollutants in wastewater. It is suggested that degradation rates be normalized to the carrier surface area, in favor of the biomass concentration, as this reflects the diffusion limitations of oxygen, and will facilitate the comparison of different biofilm systems.

Removal of pharmaceutical metabolites in wastewater ozonation including their fate in different post-treatments.

Advanced treatment technologies for the removal of pharmaceuticals and other organic micropollutants in WWTPs primarily target the removal of parent compounds. Nevertheless, the removal of metabolites originating from human- or microbial metabolism during biological treatment needs comparable consideration, as some of them might be present in high concentrations and contribute to toxicity. This study was conducted to elucidate the removal of human and microbial metabolites of pharmaceuticals as a function of the specific ozone dose. Ozonation was performed on four sites with two pilot- and two full-scale plants operated downstream of conventional activated sludge plants. The ozone reactivity of all metabolites (expressed as the ozone dose to remove 90% of the compound/decadic ozone dose) was lower than those of their parent compounds. The decadic ozone dose was 1.0, 1.3 and 1.1 mg O3/mg DOC for Epoxy-carbamazepine, Di-OH-carbamazepine and N-Desmethyl tramadol, respectively. 20-40% of the remaining metabolites were removed in a polishing sand/BAC-filter (biological activated carbon). Similar removal was observed for Epoxy-carbamazepine, Di-OH-carbamazepine and Hydroxy-diclofenac in a constructed wetland. However, the sand/anthracite filter had no effect. All four metabolites were removed in a GAC (granulated activated carbon) filter.

MBBRs as post-treatment to ozonation: Degradation of transformation products and ozone-resistant micropollutants.

The degradation potential of micropollutants and transformation products in biological post-treatment after ozonation is partly unknown. A pilot plant with ozonation and subsequent biological treatment in a moving bed biofilm reactor (MBBR) was thus operated over 16 months to investigate the removal of micropollutants and the formation and removal of N-oxide transformation products. Lab-scale kinetic experiments were performed in parallel. At a moderate ozone dose of 0.5 g O3 g-1 DOC, further degradation of gabapentin and 3 iodinated contrast media (iomeprol, iopamidol, and iohexol) could be induced by the biofilm at prolonged exposure times. To facilitate comparison of feeding regimens in biofilm systems a new surface-related degradation rate constant was introduced. The availability of substrates in the pilot MBBR influenced the micropollutant degradation kinetics with increasing and decreasing degradation rates. N-oxides from erythromycin, clarithromycin, tramadol, and venlafaxine were formed during ozonation and could not be degraded by the biofilm.

Ozonation of Sitagliptin: Removal Kinetics and Elucidation of Oxidative Transformation Products.

Due to the increasing use and high excretion rates, high quantities of the antidiabetic drug sitagliptin (STG) enter wastewater treatment plants (WWTPs). In conventional biological treatment, only a moderate removal was achieved, and thus, STG can be detected in WWTP effluents with concentrations in the higher ng/L range. Ozonation is a widely discussed technique for advanced wastewater treatment. In lab-scale experiments, STG showed pH-dependent removal kinetics with a maximum apparent rate constant of k ∼1 × 104 M-1 s-1 at pH ≥ 9. With an apparent rate constant of kO3 = (1.8 ± 0.7) × 103 M-1 s-1 at pH 8, STG can be considered to be readily degraded by ozonation of WWTP effluents. Ozone attacks the primary amine moiety of STG, leading to nitro-STG (TP 437) (the primary amine moiety is transformed into a nitro group). Furthermore, a diketone (TP 406) was formed, which can be further degraded by ozone. Lab-scale and pilot-scale experiments on ozonation of WWTP effluents confirmed that the ozone attack of STG was incomplete even at high ozone doses of 1.7 and 0.9 mg O3/mg DOC, respectively. These experiments confirmed that nitro-STG was formed as the main TP in the wastewater matrix. Two other TPs, TP 421c and TP 206b, were also detected, albeit with low intensities.

Ozone dose dependent formation and removal of ozonation products of pharmaceuticals in pilot and full-scale municipal wastewater treatment plants.

The removal of micropollutants from municipal wastewater is challenged by the number of compounds with diverse physico-chemical properties. Ozonation is increasingly used to remove micropollutants from wastewater. However, ozonation does not necessarily result in complete mineralization of the organic micropollutants but rather transforms them into new compounds which could be persistent or have adverse environmental effects. To explore ozone dose dependency of the formation and successive removal of ozonation products, two pilot-scale and one full-scale ozonation plants were operated subsequent to a conventional activated sludge treatment. The results from these trials indicated that the concentrations of several N-oxides, such as Erythromycin N-oxide, Venlafaxine N-oxide and Tramadol N-oxide, increased up to an ozone dose of 0.56-0.61 mg O3/mg DOC while they decreased at elevated doses of 0.7-1.0 mg O3/mg DOC. Similar results were also obtained for two transformation products of Diclofenac (Diclofenac 2,5-quinone imine and 1-(2,6-dichlorophenyl)indolin-2,3-dione) and one transformation product of Carbamazepine (1-(2-benzoic acid)-(1H,3H)-quinazoline-2,4-dione), where the highest concentrations appeared around 0.27-0.31 mg O3/mg DOC. The formation maximum of a given compound occurred at a specific ozone dose that is characteristic for each compound, but seemed to be independent of the wastewater used for the experiments at the two pilots and the full-scale plant.

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.

Nontarget analysis: A new tool for the evaluation of wastewater processes.

Strategies to determine the removal efficiency of micropollutants in wastewater treatment plants (WWTPs) are widely discussed. Especially the evaluation of the potential benefit of further advanced treatment steps such as an additional tertiary treatment based on ozonation or activated carbon have come into focus. Such evaluation strategies are often based on the removal behavior of known micropollutants via target or suspected analysis. The utilization of nontarget analysis is considered to lead to a more comprehensive picture as also unknown or not expected micropollutants are analyzed. Here, the results of an evaluation via target and nontarget analysis were compared for biological treatment (BT) processes of eleven full-scale WWTPs and three different post-treatments (PTs): one sand filter (SF) and two granular activated carbon (GAC) filters. The similarity of the determined removals from target and nontarget analysis of the BTs increased significantly by excluding easily degradable "features" from the nontarget evaluation. A similar ranking of the removal trends for the BTs could also be achieved by comparing this new subset of nontarget features with a set of nine readily to moderately biodegradable micropollutants. This observation suggests that a performance ranking of BTs based either on target or nontarget analysis is plausible. In contrast to the BTs, the evaluation of the three PTs revealed that the difference of feature removal between SF and the two GACs was small, but large for the target analytes with substantially higher removal effciencies for the GACs compared to the SF. In addition to the removal behavior, the nontarget analysis provided further information about the number and quantity of transformation products (TPs) in the effluent from the BTs. For all BTs more than half (55-67%) of the features detected in the effluent were not found in the influent. A comparable proportion of TPs was also detected after GAC and sand filtration due to their microbial activities.

PAC dosing to an MBBR - Effects on adsorption of micropollutants, nitrification and microbial community.

Two nitrifying MBBR reactors were operated in parallel, one with PAC dosing and one without, to determine the effects of PAC dosing on nitrification and micropollutant adsorption in municipal wastewater. The removal of micropollutants was evaluated for several doses of PAC and batch experiments were performed to measure adsorption kinetics and nitrification rates. The influence of PAC on the nitrifying microbial community was examined by high-throughput amplicon sequencing. Long-term operation of the pilot reactors showed that nitrification could be maintained while supplying PAC at increasing doses, as confirmed by high nitrification rates and significant abundance of nitrifying bacteria. The adsorption of organic micropollutants could be controlled by the PAC dose, and increased dosing resulted in corresponding improvements in removal efficiency. Biomass, suspended or attached to carriers, did not interfere with the adsorption of organic micropollutants. Freundlich isotherms obtained from the batch experiments were used to predict removal of organic micropollutants in the pilot reactors, suggesting that batch adsorption experiments can be used to predict micropollutant removal on a full scale. Collectively, the results show that nitrification and adsorption of organic micropollutants can be performed simultaneously in an MBBR.

Impact of activated sludge ozonation on filamentous bacteria viability and possible added benefits.

Ozone was applied to return activated sludge in full-scale to study how ozone impacts filamentous bacteria viability (Live/Dead®). Additionally, the ozonated sludges were subjected to anaerobic digestion trials and analysis of micropollutants (MPs). Ozone treatment (3-4.8 g O3/kg total suspended solids) improved the settling properties of the sludge by lowering the diluted sludge volume with 7-35%. Ozone inactivated filamentous bacteria outside the floc structures and the fraction of inactivated filaments increased with an increasing ozone dose. It was observed that ozone treatment may act selectively towards different types of filaments. With respect to the two dominating morphotypes present, Type 0041 filamentous bacteria were found to be more resistant to ozone attack than Microthrix parvicella. Thus, higher ozone doses may be required to mitigate sludge bulking caused by Type 0041 filaments. No effects could be discerned by ozone addition on neither the methane production of the sludge nor on the concentrations of MPs analysed for this study. The lack of effect on both methane production and micropollutant removal was deemed to be caused by insufficient ozone doses.