Feeding composition and sludge retention time both affect (co-)metabolic biotransformation of pharmaceutical compounds in activated sludge systems

Feeding composition and sludge retention time both affect (co-)metabolic biotransformation of pharmaceutical compounds in activated sludge systems Lorena Gonzalez-Gil, Eduardo Fernandez-Fontaina, Randolph R. Singh, Juan M. Lema, Marta Carballa, Diana S. Agac Accepted Manuscript How to cite: Journal of Environmental Chemical Engineering, 9 (2021), 105123 https://doi.org/10.1016/j.jece.2021.105123 Copyright information: © 2021 Elsevier Ltd. This manuscript version is made available under the CC-BY-NC-ND 4.0 license (http://creativecommons.org/licenses/by-nc-nd/4.0/) Graphical Abstract 1 Feeding composition and sludge retention time both affect (co-)metabolic biotransformation of pharmaceutical compounds in activated sludge systems Lorena Gonzalez-Gil1,2ǂ, Eduardo Fernandez-Fontaina1,3ǂ, Randolph R. Singh3, Juan M. Lema1, Marta Carballa1* and Diana S. Aga3 1CRETUS Institute, Department of Chemical Engineering, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain 2Defence University Centre, Spanish Naval Academy, Plaza de España, 36920, Marín, Spain 3Department of Chemistry, University at Buffalo, The State University of New York, Buffalo, NY, 14260, United States ǂ Both authors have equally contributed to this work *Corresponding author: Marta Carballa ([email protected]) Highlights (for review) 2 HIGHLIGHTS The role of heterotrophs and nitrifiers on the (co-)metabolism of PhACs was explored. Longer SRT-sludge has a higher capacity to biotransform most of the selected PhACs. Feeding composition affects biological activities and thus the co-metabolism of PhACs. Slow-growing heterotrophs are crucial for the biotransformation of some PhACs. Acetaminophen, caffeine, and iopromide could be growth substrates of heterotrophs. 1 Feeding composition and sludge retention time both affect (co-)metabolic biotransformation of pharmaceutical compounds in activated sludge systems Lorena Gonzalez-Gil1,2ǂ, Eduardo Fernandez-Fontaina1,3ǂ, Randolph R. Singh3, Juan M. Lema1, Marta Carballa1* and Diana S. Aga3 1CRETUS Institute, Department of Chemical Engineering, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain 2Defence University Centre, Spanish Naval Academy, Plaza de España, 36920, Marín, Spain 3Department of Chemistry, University at Buffalo, The State University of New York, Buffalo, NY, 14260, United States ǂ Both authors have equally contributed to this work *Corresponding author: Marta Carballa ([email protected]) Revised Manuscript (Unmarked version) 2 GRAPHICAL ABSTRACT HIGHLIGHTS The role of heterotrophs and nitrifiers on the (co-)metabolism of PhACs was explored. Longer SRT-sludge has a higher capacity to biotransform most of the selected PhACs. Feeding composition affects biological activities and thus the co-metabolism of PhACs. Slow-growing heterotrophs are crucial for the biotransformation of some PhACs. Acetaminophen, caffeine, and iopromide could be growth substrates of heterotrophs. 3 ABSTRACT The role of heterotrophic and nitrifying microorganisms in the (co-)metabolic biotransformation of 10 pharmaceutically active compounds (PhACs) was investigated. To this aim, biotransformation assays were performed with heterotrophic and nitrifying sludge developed separately in a two-stage full-scale activated sludge system. Each stage was operated at different inflow wastewater characteristics and sludge retention times (on average 8 d and 35 d). The biotransformation capacity of each sludge was evaluated in the absence of primary substrate and in the presence of acetate and ammonium, to independently elucidate the co-metabolic role of heterotrophs and nitrifiers present in both sludges. Trimethoprim, diclofenac and carbamazepine were recalcitrant (removal < 5% after 1 d; biotransformation rate < 50 µg/g VSS·d) under all the tested conditions. High concentrations of caffeine, acetaminophen and iopromide were quickly biotransformed (> 80% after 1 d; > 800 µg/g VSS·d) in the absence of primary substrates. The heterotrophic sludge only showed a cometabolic effect towards erythromycin, which increased its biotransformation rate between 43-53% when acetate and ammonium were supplied. In contrast, when stimulated, nitrifiers and slow-growing heterotrophs present in the nitrifying sludge co-metabolically biotransformed acetaminophen, ibuprofen and naproxen to a significant extent. Sulfamethoxazole was recalcitrant, except when the nitrifying sludge was fed with acetate (> 800 µg/g VSS·d), suggesting that slow-growing heterotrophs co-metabolically biotransformed it. This study provides evidence that biotransformation of PhACs depends on several metabolic activities, as the heterotrophic activity of the nitrifying sludge, which are not only determined by the SRT but also by the feeding composition. KEYWORDS: biodegradation; heterotrophs; cometabolism; nitrifiers; organic micropollutants; wastewater treatment plant. 4 1. INTRODUCTION Organic micropollutants, such as pharmaceutically active compounds (PhACs), are continuously being released into the environment from effluents of wastewater treatment plants (WWTPs) (Tran et al., 2018). Despite their relatively low concentrations, these compounds pose deleterious ecological effects. For instance, the persistence of antibiotics in the environment has been linked to the emergence of antibiotic-resistant bacteria and the spread of resistance genes in the environment (Pruden et al., 2013). The presence of these pollutants in drinking water sources is also of concern and concentration limits will likely be established in the near future (EC, 2018; EPA, 2016). Consequently, their release through WWTP effluents will be also restrained. Significant research efforts have been conducted to tackle the removal of organic micropollutants in WWTPs. The identification and isolation of different bacterial strains capable of degrading several organic micropollutants co-metabolically in the presence of primary substrates (Larcher and Yargeau, 2011; Almeida et al., 2013; Liu et al., 2013; Zhou et al., 2013; Men et al., 2017) or metabolically, as growth substrate (De Gusseme et al., 2011; Summers et al., 2012; Zhang et al., 2013), suggest that the composition of microbial communities in WWTPs can influence the pathway and extent of micropollutant biotransformation. However, the underlying biological mechanisms (metabolism or cometabolism) and the driving factors behind the biotransformation of most organic micropollutants in activated sludge reactors are still unclear (Fischer and Majewsky, 2014; Nsenga and Meng, 2019), hampering the development of models to predict their removal efficiencies and concentrations in WWTP effluents (Tran et al., 2013). Several studies have investigated the influence of operational parameters and the type of biomass on the biotransformation of micropollutants in activated sludge systems (Suárez et al., 2010; Khunjar et al., 2011; Fernandez-Fontaina et al., 2012; Achermann et al., 2018; 5 Lakshminarasimman et al., 2018; Tran et al., 2018; Kennes-Veiga et al., 2021). Yet, most of the differences found in their removal efficiency remain unexplained (Tran et al., 2018). Part of these variabilities has been attributed to the change of the sludge retention time (SRT). Operating activated sludge reactors at longer SRT (Vieno et al., 2007; Suarez et al., 2010) results in more diverse bacterial populations and a higher proportion of autotrophic ammonia oxidizing microorganisms (AOMs) due to their low specific growth rates. Along with the SRT, the feeding composition (e.g., C:N ratio and degradability of the carbon source) and the hydraulic retention time (HRT) are key for the growth of the different microbial populations, as they determine organic and nitrogen loading rates, and consequently the relative composition of heterotrophs and nitrifiers in activated sludge (Gallé et al., 2019; Nguyen et al., 2021). In addition, the type of growth substrate may alter the catabolic enzymes expressed by a community, thus influencing the metabolic and co-metabolic capacity to biotransform PhACs (Nguyen et al., 2021). Nevertheless, the effect of the feeding composition is by far the less unexplored parameter in activated sludge systems. Nitrifying conditions (long SRT and low C:N ratio) seem to be more effective than heterotrophic conditions (short SRT and high C:N ratio) for the biotransformation of some micropollutants (Suarez et al., 2010; Fernandez-Fontaina et al., 2012), particularly those that undergo oxidative transformations (Helbling et al., 2012). Although most studies associate biotransformation with the main metabolic activity of the sludge, according to some authors, the higher removal rates observed at high SRT cannot be strictly linked to the action of nitrifiers (Batt et al., 2006; Fernandez-Fontaina et al., 2012; Men et al., 2017; Achermann et al., 2018). Actually, some studies suggest that improved removal of PhACs in nitrifying reactors is not due to the presence of nitrifiers but to the expansion in the composition of the heterotrophic bacterial community developed (Falås et al., 2012; Fernandez-Fontaina et al., 2016). This is consistent with the ecological theory, which postulates that greater diversity 12 results, specific biotransformation rates (rsb, µg PhAC/gVSS·d) were preferred in this study (Eq.2). 𝑟𝑠𝑏 =𝑟𝑏 𝑋𝑉𝑆𝑆 (2) Significant differences (p ≤ 0.05) between two specific biotransformation rates were calculated by the Student’s t-test (Andrade and Estevez-Perez, 2014). 3. RESULTS AND DISCUSSION 3.1 Heterotrophic and nitrifying activities 3.1.1 Biological activities of the sludge The respirometry tests performed to determine the biological activities of the two types of activated sludge from the Amherst WWTP (Table 1) confirmed that the biomass collected from stage 1 (AS1, SRT = 8 d) was predominantly heterotrophic (727 ± 62 mg O2/g VSS·d), displaying very low nitrifying activity (9 ± 1 mg N-NH4+/g VSS·d). On the contrary, nitrifying activity was much higher (117 ± 12 mg N-NH4+/g VSS·d) in the activated sludge from stage 2 (AS2, SRT = 35 d) with lower heterotrophic activity (152 ± 71 mg O2/g VSS·d). Heterotrophs in AS2 are responsible for degrading part of the recalcitrant organic matter not consumed by AS1 in stage 1. Therefore, as expected, the difference in the sludge age and feeding characteristics lead to different microbial compositions and activities in the activated sludge from bioreactors of stage 1 and 2. Table 1. Operational conditions in Stage 1 (organic matter degradation) and Stage 2 (nitrification) of the biological treatment of Amherst WWTP and metabolic activities of the activated sludge from each stage (AS1 and AS2, respectively) determined by the respirometry tests detailed in section 2.2. 13 Heterotrophic sludge (AS1) Nitrifying sludge (AS2) Operational conditions HRT (h) 1 2 SRT (d) 8 35 Microbial activities Heterotrophic (mg O2/g VSS·d) 727 ± 62 152 ± 71 Nitrifying (mg N-NH4+/g VSS·d) 10 ± 1 117 ± 12 Endogenous (mg O2/g VSS·d) 232 ± 27 72 ± 30 3.1.2 Heterotrophic and nitrifying activities in the biotransformation assays Heterotrophic and nitrifying populations in the two types of sludge (AS1 and AS2) were preferentially activated by feeding acetate and ammonium during the biotransformation experiments with PhACs. The specific activities along the six biotransformation assays were assessed in terms of Organic Removal Rate (ORR) and Nitrification Rate (NR) (Fig. 1). More details about the concentrations of ammonium, nitrite, nitrate and dissolved organic carbon are displayed in Fig. S2 and Fig. S3 of the Supplementary Information. As expected, the heterotrophic sludge developed at low SRT (AS1) reached the highest ORR (665 mg COD/g VSS·d) in the experiment fed with acetate (heterotrophic conditions), which corresponds to 72% removal of the total organic loading rate applied (921 mg COD/g VSS·d) (Fig. 1). As shown in Fig. S2, this effectiveness is higher at the beginning of the experiment when remaining nitrogen from the sludge is present. Yet, despite supplying extra nitrogen could increase the heterotrophic activity, it could also enhance nitrifiers, and thus hinder our objective. Consequently, a minor NR was achieved in the experiments performed with AS1; only a slight increase is observed when ammonium was supplied. The nitrifying sludge developed at high SRT (AS2) showed a clear maximum of ORR (125 mg COD/g VSS·d) under heterotrophic conditions (acetate supply), corresponding to an organic matter removal 14 efficiency of 83%, and a maximum NR of 37 mg N/g VSS·d under nitrifying conditions, one order of magnitude higher than that achieved with AS1. Fig. 1. Average organic removal rate (ORR) and nitrification rate (NR) in the biotransformation experiments performed with heterotrophic (AS1) and nitrifying sludge (AS2) under different feeding conditions: acetate supply (activation of heterotrophs), ammonium supply (activation of nitrifiers) and no substrate addition (control). Prior to the biotransformation experiments, AS1 and AS2 were aerated for 48 h to allow complete removal of residual substrates. The remaining concentrations of organic matter and ammonium nitrogen after this aeration period were very low: 7.2 mg N-NH4+/L and 22.1 mg DOC/L in AS1 and ˂0.01 mg N-NH4+/L and 9.7 mg DOC/L in AS2. Therefore, although almost no readily biodegradable substrate was present in the control experiments, microbial lysis released organic matter and ammonium nitrogen (Fig. S2 and Fig. S3), which could explain the low but still noticeable ORR and NR of the control assays (Fig. 1). Similarly, the heterotrophic activity observed under nitrifying conditions (no organic carbon was supplied) 0 8 16 24 32 40 0 150 300 450 600 750 Acetate supply Ammonium supply Control NR (mg N/g VSS·d) ORR (mg O2/g VSS·d) Heterotrophic sludge (AS1) 0 8 16 24 32 40 0 150 300 450 600 750 Acetate supply Ammonium supply Control NR (mg N/g VSS·d) ORR (mg O2/g VSS·d) Nitrifying sludge (AS2) 15 and the nitrifying activity detected under heterotrophic conditions (no nitrogen was supplied) with AS1 and AS2 (Fig. 1) might be mostly due to microbial lysis. Finally, it should be highlighted that the main biological activities of AS1 and AS2 were very similar in the control assays performed with and without PhACs. According to bibliographic data, it was not expected a substantial inhibitory effect of the selected PhAC concentrations in the activated sludge activities (Dokianakis et al., 2004; Wang and Gunsch et al., 2011; Katipoglu-Yazan et al., 2013; Katsou et al., 2016). According to the previous results (Table 1 and Fig. 1), the control assays with PhACs will be useful for elucidating the metabolic or co-metabolic biotransformation capacity of a heterotrophic sludge (AS1) versus a nitrifying sludge (AS2) at low biological activities. Moreover, Fig. 1 reveals that the main metabolic activity of AS1 (heterotrophic) and AS2 (nitrifying) was clearly promoted by feeding acetate and ammonium, respectively. Therefore, the comparison between the biotransformation results obtained in these assays with the control will provide insights on the heterotrophic and nitrifying co-metabolism of the selected PhACs. Moreover, it was demonstrated that heterotrophs are also present in AS2 and nitrifying bacteria in AS1 (Table 1 and Fig. 1); thus, the co-metabolic role of these secondary sludge activities on the biotransformation of PhACs could be investigated in the experiments with acetate and ammonium supply to AS2 and AS1, respectively. 3.2 Biotransformation of PhACs by the heterotrophic and nitrifying sludge Fig. 2 shows the specific biotransformation rates of the 10 selected PhACs in the control assays (no feeding) performed with the heterotrophic sludge (AS1) and the nitrifying sludge (AS2). According to their behaviour, the PhACs can be classified into three groups: (i) compounds quickly biotransformed (> 800 µg/g VSS·d and removal > 80% after 1 d) at least in one of the sludges, i.e., ACM, CAF, and IOP; (ii) compounds slowly biotransformed (100400 µg/g VSS·d and removal 10-40% after 1 d) at least in one of the sludges, i.e., IBP, ERY 16 and NPX; and (iii) persistent PhACs (< 50 µg/g VSS·d and removal < 5% after 1 d) in both activated sludges, i.e., SMX, CBZ, DCF and TMP. Fig. 2. Biotransformation rate of PhACs attained by the heterotrophic (AS1) and nitrifying (AS2) activated sludge in the control experiments (no addition of primary substrate). Error bars represent standard error values of the biotransformation rate calculated considering 10-11 experimental points for AS1 and 9 experimental points for AS2. The asterisks highlight those PhACs whose biotransformation rates (with AS1 and AS2) are significantly different (p ≤ 0.05). 3.2.1 Biotransformed PhACs Overall, the biotransformation results obtained in this study for PhACs of group (i) and group (ii) agree with those reported in real WWTPs (Tran et al., 2018). In fact, among the 60 emerging micropollutants reviewed by Tran et al. (2018), ACM followed by CAF, presented the highest biodegradation kinetic constant. In the case of IBP, it varied among the moderatehigh range, and the biotransformation kinetics of IOP, ERY and NPX was considered moderate, although IOP often achieved high removal efficiencies. As shown in Fig. 2, except for CAF and NPX, the biotransformation rate of group (i) or group (ii) of PhACs depend on the sludge type (differences were statistically significant according to Student’s t-test; p ≤ 0.05); that is to say, they were affected by the microbial 1 10 100 1000 10000 ACM * CAF IOP * IBP * ERY * NPX SMX * CBZ DCF TMP * Biotransformation rate (µg/g VSS·d) Heterotrophic sludge (AS1) Nitrifying sludge (AS2) (iii) Persistent (ii) Slowly biotransformed(i) Quickly biotransformed 17 composition of the biomass. The biotransformation rates achieved by the heterotrophic sludge (AS1) were significantly higher than those obtained by the nitrifying sludge (AS2) in the case of ACM and ERY; in fact, ERY was not removed at all by AS2. On the contrary, quicker biotransformation in the control experiment performed with AS2 was observed for IBP and IOP, which was hardly removed by AS1. Therefore, depending on the compound, biotransformation could be favoured either under heterotrophic or nitrifying conditions. However, as previously mentioned, the differences among AS1 and AS2 are not only due to the SRT but also to the differences in the feeding characteristics (AS2 is fed with the resulting effluent treated with AS1), which might also affect the microbial composition and the enzymatic activities. Consequently, the effects observed on the biotransformation of PhACs cannot be directly linked to the SRT. In fact, the relationship between SRT and biotransformation is still a controversial topic in the literature. For instance, a positive correlation between longer SRT and better biotransformation of PhACs has been reported for IBP (Clara et al., 2005), NPX (Suarez et al., 2010; Falås et al., 2012), and IOP (Batt et al., 2006). However, the opposite trend, where shorter SRT resulted in higher biotransformation, was also reported for IBP (Vuono et al., 2016; Gallé et al., 2019), NPX and CAF (Vuono et al., 2016). These discrepancies in the literature suggest that the SRT is not the only governing factor that should be considered in delineating the microorganisms responsible for the biotransformation of PhACs (Gallé et al., 2019). Other factors, such as substrate composition, might change the metabolic activities of the microbial community, and thus their biotransformation capacity (Fischer and Majewsky, 2014). 3.2.2 Persistent PhACs Although some significant differences were found in the biotransformation rates (Fig. 2) of PhACs from group (iii), the values were very low and almost no biotransformation (< 5% after 1 d) was observed. Therefore, it can be stated that all PhACs from group (iii) presented a 18 similar behaviour with both types of activated sludge. Moreover, as shown in Table 2, except for SMX which will be further discussed in the next section, CBZ, DCF and TMP were also recalcitrant under all tested conditions. The recalcitrance of these three compounds is widely supported in the literature. For instance, the anti-epileptic drug CBZ proved to be very persistent in WWTPs and the environment (Onesios et al., 2009; Tran et al., 2018). Even at long SRTs, obtained in biological treatments designed for nutrient removal or in membrane bioreactors, no biotransformation of CBZ has been observed (Radjenovic et al., 2009; Suarez et al., 2010; Fernandez-Fontaina et al., 2012). Regarding the anti-inflammatory drug DCF, the reported removal efficiencies support its low biotransformation potential in conventional activated sludge systems (Joss et al., 2006; Nguyen et al., 2019) and throughout WWTPs (Gallé et al., 2019). However, in some particular circumstances, the biotransformation of DCF seems to be improved; for instance, at long SRTs in membrane bioreactors (Vieno and Sillanpää, 2014), and under nitrifying conditions (Suarez et al., 2010; Fernandez-Fontaina et al., 2012). These results cannot be generalized, since other authors found opposite trends. According to Gallé et al. (2019), the removal of DCF seems to be quite independent of operational parameters. Likewise, although the antibiotic TMP has been usually regarded as difficult to remove in activated sludge systems (Verlicchi et al., 2012) and throughout WWTPs (Onesios et al., 2009), some studies have obtained relevant biotransformation efficiencies at diverse conditions: high nitrifying activities (Fernandez-Fontaina et al., 2012; Men et al., 2017); heterotrophic conditions (Khunjar et al., 2011); a membrane bioreactor with high SRT (Göbel et al. 2007) and an activated sludge system with low SRT and phosphorus accumulating organisms (Lakshminarasimman et al., 2018); Our results, and the comparison with other studies, confirm that neither heterotrophic nor nitrifying activities may improve the biotransformation of some PhACs. This behaviour is 19 observed when the PhACs have a highly stable chemical structure (e.g., CBZ), or when their biotransformation depends on physiological capabilities and/or microorganisms not limited by the SRT or the feeding composition (e.g., DCF and TMP). 3.3 Stimulation of the co-metabolic biotransformation of PhACs by heterotrophs and nitrifiers The results obtained with the control assays (Fig. 2) are not enough to attribute the biotransformation of PhACs neither to heterotrophs (the main metabolic activity of AS1) nor to nitrifiers (the main metabolic activity of AS2), since both populations are present in AS1 and AS2 (Fig. 1). Moreover, the biotransformation mechanisms (i.e., metabolism or cometabolism) behind the removal of the selected PhACs remain unknown. Therefore, to clarify the role of heterotrophs and nitrifiers, their co-metabolic activities were promoted by separately feeding both activated sludges with acetate and ammonium (Table S1). It should be noted that a co-metabolic effect implies an increase in the biotransformation rate of PhACs when the metabolic (enzymatic) activity of the biomass is promoted by adding a specific growth substrate. However, if this effect is not observed, co-metabolic biotransformation cannot be ruled out, since the reaction rate increases linearly with the enzymatic activity, but just until a threshold concentration of enzyme (Frey and Hegeman, 2007; Purich, 2010). Additionally, the biotransformation rate of a compound is not only limited by the metabolic activity of the biomass, but also by other factors, such as reversibility of biological reactions (Gonzalez-Gil et al., 2018; Gonzalez-Gil et al., 2019). Following the classification of PhACs made in Fig. 2, the fate of compounds belonging to group (i), (ii) and (iii) in all the biotransformation assays is depicted in Fig. 3, Fig. 4 and Fig. 5/Fig. S6, respectively. The corresponding biotransformation rates are summarized in Table 2. By comparing the biotransformation results obtained in the control assays (no primary substrate) with the experiments fed with acetate or ammonium, a significant (p ≤ 0.05) co- 20 metabolic effect (concomitant increase in the biotransformation rate and biological activity) was observed for ACM, IBP, NPX and SMX with AS2, and for ERY with AS1. Table 2. Biotransformation rates (µg/g VSS·d) of PhACs by the heterotrophic (AS1) and nitrifying (AS2) sludge in the control assays and under heterotrophic and nitrifying conditions. Biotransformation rate (µg/g VSS·d) Heterotrophic sludge (AS1) Nitrifying sludge (AS2) Control Heterotrophic conditions Nitrifying conditions Control Heterotrophic conditions Nitrifying conditions ACM 5202 ± 200 4656 ± 400 5102 ± 400 852 ± 80 1950 ± 130 1837 ± 70 CAF 860 ± 30 478 ± 40 840 ± 50 866 ± 60 1004 ± 70 458 ± 50 IOP 105 ± 20 80 ± 6 87 ± 10 3647 ± 1000 1627 ± 500 1210 ± 300 IBP 212 ± 30 150 ± 30 192 ± 30 389 ± 60 2006 ± 300 1921 ± 100 ERY 195 ± 10 280 ± 30 298 ± 20 15 ± 10 62 ± 20 93 ± 30 NPX 99 ± 10 44 ± 9 81 ± 7 66 ± 20 616 ± 30 363 ± 80 SMX 25 ± 10 38 ± 10 29 ± 9 – 858* ± 300 16 ± 10 CBZ 28 ± 10 36 ± 10 37 ± 10 7 ± 5 7 ± 7 9 ± 9 DCF – 3 ± 9 1 ± 6 11 ± 10 11 ± 10 31 ± 10 TMP 44 ± 10 55 ± 10 48 ± 10 – – – *Biotransformation rate was calculated after the 2-days lag phase observed in Fig. 8. 3.3.1 PhACs quickly biotransformed As depicted in Fig. 3, among the compounds in group (i), the supply of a growth substrate (acetate or ammonium) only exhorted a significantly positive effect in the biotransformation rate of ACM with AS2, which raised from 852 µg/g VSS·d in the control up to 1950 µg/g VSS·d in the presence of acetate. Oppositely, a co-metabolic effect was not observed for ACM in the assays with AS1, and neither for CAF nor for IOP in the assays with AS1 and AS2. The lack of a co-metabolic effect in combination with the fast biotransformation rates attained in the control assays, make plausible the usage of these compounds as carbon and energy sources by bacteria present in AS1 and AS2. Although more detailed studies are needed to support this hypothesis, bacterial strains capable of using ACM as sole carbon, nitrogen and energy source have been isolated from activated sludge in several 21 studies (De Gusseme et al., 2011; Zhang et al., 2013). Regarding CAF, its quick disappearance was followed by the formation of the N-demethylated metabolites paraxanthine and theobromine, which in turn biotransformed later (Fig. S4). Although the TPs of paraxanthine and theobromine were not identified, further N-demethylation of these metabolites is possible leading to the formation of xanthine and formaldehyde that can enter metabolic pathways in bacteria (Summers et al., 2012). A similar pattern in the formation of both metabolites was observed under all tested conditions; hence, the same biochemical mechanism is responsible for the biotransformation of CAF in activated sludge, regardless of the sludge characteristics and biological activities. Based on all these experimental observations, it is reasonable to assume that ACM, CAF and IOP were biotransformed via direct metabolism. However, the role of co-metabolic biotransformation cannot be ignored because organic carbon and nitrogen are also available in the control assays due to microbial cell lysis (Fig. S2). Yet, the low co-metabolic activities under these conditions might not explain their fast biotransformation. 28 Despite O-demethylation being a common biotransformation pathway in activated sludge systems (Quintana et al., 2005), the presence of the corresponding TP (O-desmethylnaproxen) was not observed in our experiments. 3.3.3 Persistent PhACs The persistence of CBZ, DCF and TMP was not modified by either promoting the heterotrophic or nitrifying activities (Fig. S6). Surprisingly, the antibiotic SMX, which was persistent in both control assays (Fig. 2), was completely biotransformed when heterotrophs from AS2 (SRT = 35 d) were fed with acetate after a lag-phase of 2 days (Fig. 5) (differences with the control were statistically significant according to Student’s t-test; p ≤ 0.05). However, no significant (p > 0.05) removal was observed when acetate was supplied to AS1 (Fig. 5), which had a 4-fold higher heterotrophic activity (Fig 2). Therefore, it can be concluded that SMX co-metabolic biotransformation is not dependent on heterotrophic activity in general, but on slow-growing heterotrophic activities, as recently demonstrated by Peng et al. (2019) in an activated sludge reactor with heterotrophic activity and a long SRT (30 d). Likewise, Fernandez-Fontaina et al. (2016) found that stimulating the heterotrophic activity present in nitrifying activated sludge enhances the biotransformation of SMX. Additionally, while low removal efficiencies (< 20%) of SMX were obtained by Joss et al. (2006) with conventional activated sludge, higher biotransformation rates were achieved in membrane bioreactors with characteristic long SRTs (Radjenovic et al., 2009). In contrast, other authors, such as Zhou et al. (2019), also reported biotransformation of SMX by ammonia oxidizers. 29 Fig. 5. Fate of SMX in the control assays (no additional substrate), under heterotrophic conditions (acetate supply) and under nitrifying conditions (ammonium supply) performed with AS1 and AS2. 4. CONCLUSIONS The strategy followed in this study allowed to stimulate the main and the secondary heterotrophic and nitrifying activities present in both a heterotrophic (developed at low SRT and high C:N ratios) and a nitrifying (developed at high SRT and low C:N ratios) sludge, by changing the feeding characteristics. This novel approach provided new insights about the metabolic and co-metabolic role of heterotrophs and nitrifiers in the biotransformation of some PhACs in activated sludge systems. Moreover, it was an appropriate strategy to elucidate if biotransformation of PhACs was conducted by the main or secondary activities of sludge, which is still a disregarded topic in literature. In summary, CBZ, DCF and TMP were recalcitrant under all conditions tested, hence their biotransformation seems to not depend on the heterotrophic or nitrifying activity. The heterotrophic sludge (AS1) favoured the biotransformation of ACM and ERY, while the nitrifying sludge (AS2) presented a higher capacity to biotransform IOP, NPX, IBP and SMX. Therefore, it seems that AS2 possess an enhanced degradation capability in comparison to AS1. The latter cannot only be attributed to the role of nitrifiers, but also to the slow-growing heterotrophic microorganisms. Consequently, our results highlight that the main metabolic 0.0 0.2 0.4 0.6 0.8 1.0 1.2 012345 C/C0 Time (d) Sulfamethoxazole 0.0 0.2 0.4 0.6 0.8 1.0 1.2 0 1 2 3 4 5 C/C0 Time (d) Sulfamethoxazole Acetate supply Ammonium supply Control Heterotrophic sludge (AS1) Nitrifying sludge (AS2) 30 activity of the sludge might not be responsible for the co-metabolic biotransformation of PhACs. Overall, this study demonstrates that not only the SRT, but also the feeding composition exerts a huge influence on the microbial composition and biological activities of the activated sludge and, consequently, on its capability to biotransform PhACs. Future research should focus on the identification of the specific slow-growing heterotrophic strains that biotransform PhACs and the factors that promote their growth, to optimize the performance of WWTPs in terms of organic micropollutant removal. 5. ACKNOWLEDGMENTS This research was supported by the UB RENEW (Research and Education in eNergy, Environment and Water) grant, the Spanish Ministry of Economy and Competitiveness COMETT Project (CTQ2016-80847-R). Authors from Universidade de Santiago de Compostela belong to CRETUS Strategic Partnership (ED431E 2018/01) and to the Galicia Competitive Research Group (GRC ED431C 2017/29), programs co-funded by FEDER (EU) and Xunta de Galicia. 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E. rRNA Gene Expression of Abundant and Rare Activated-Sludge Microorganisms and Growth Rate Induced Micropollutant Removal. Environ. Sci. Technol. 2016, 50, 6299−6309. 6 Table S4 (cont.). Details about the LC-MS/MS method employed for the detection and quantification of the selected PhACs and their metabolites. Analyte Type Retention time (min) MRM transitions Collision energy (eV) Instrument Quantitation limit (µg/L) Quantifying Qualifying Quantifying Qualifying Iopromide (IOP) Parent compound 2.8 792  573 792  559 33 39 4.00 TP819 IOP metabolite 2.4 820  587 820  714 35 29 ND TP817A IOP metabolite 2.4 818  701 818  713 27 27 ND TP805A IOP metabolite 2.2 806  559 806  686 39 31 ND TP805B IOP metabolite 2.2 806  573 806  701 33 27 ND TP787A IOP metabolite - 788  671 788  712 27 25 ND TP759 IOP metabolite - 760  671 760  684 23 23 ND TP731A IOP metabolite - 732  613 732  453 27 39 ND TP731B IOP metabolite - 732  626 732  467 23 41 ND TP729A IOP metabolite - 729  613 730  457 27 41 ND TP701A IOP metabolite - 702  613 702  454 25 43 ND TP701B IOP metabolite - 702  627 702  468 17 37 ND TP643 IOP metabolite - 644  517 644  613 19 21 ND Naproxen (NPX) Parent compound 14.8 231  185 231 – 115 10 60 0.11 O-desmethylnaproxen NPX metabolite - 215  171 - 10 ND Sulfamethoxazole (SMX) Parent compound 11.8 254  108 254  92 22 25 0.03 Trimethoprim (TMP) Parent compound 8.3 291  230 291  123 22 22 0.01 d9-Trimethoprim Internal standard 8.3 300  234 300  123 25 25 ND MRM – multiple reaction monitoring; ND – not determined because there are no commercially available standards for metabolites 7 Fig. S2. Concentrations of ammonium ( ), nitrite ( ), nitrate ( ) and dissolved organic carbon ( ) during the batch experiments performed with the heterotrophic sludge (AS1) developed in stage 1 of the Amherst WWTP. Heterotrophs were stimulated by feeding acetate (↓), nitrifiers by supplying ammonium and bicarbonate (↓) and control conditions were maintained without the addition of any primary substrate. It should be noted that ammonium and dissolved organic carbon were usually measured just before adding the corresponding substrate, hence a peak in the concentration is not observed below the arrows. Heterotrophic conditions Nitrifying conditions Control 0 100 200 300 400 500 600 0 5 10 15 20 25 30 0 1 2 3 4 5 mg C/L mg N/L t (d) 0 5 10 15 20 25 30 35 40 0 10 20 30 40 50 60 70 0 1 2 3 4 5 mg C/L mg N/L t (d) 0 5 10 15 20 25 30 35 40 0 5 10 15 20 25 30 35 40 0 1 2 3 4 5 mg C/L mg N/L t (d) 8 Heterotrophic conditions Nitrifying conditions Control Fig. S3. Concentrations of ammonium ( ), nitrite ( ), nitrate ( ) and dissolved organic carbon ( ) during the batch experiments performed with the nitrifying sludge (AS2) developed in stage 2 of the Amherst WWTP. Heterotrophs were stimulated by feeding acetate (↓), nitrifiers by supplying ammonium and bicarbonate (↓) and control conditions were maintained without the addition of any primary substrate. It should be noted that ammonium and dissolved organic carbon were usually measured just before adding the corresponding substrate, hence a peak in the concentration is not observed below the arrows. 0 5 10 15 20 25 30 35 40 0 5 10 15 20 25 30 0 1 2 3 4 5 mg C/L mg N/L t (d) 0 5 10 15 20 25 30 35 40 0 5 10 15 20 25 30 0 1 2 3 4 5 mg C/L mg N/L t (d) 9 Fig. S4. Biotransformation pathway of caffeine and peak area ratios referred to the internal standard (d10-CBZ) of caffeine (in the left axis), and its formed metabolites, paraxanthine and theobromine (in the right axis), determined by LC-MS/MS under multiple reaction monitoring (MRM) for the batch experiments performed with AS1 and AS2 under heterotrophic (acetate supply), nitrifying (ammonium supply) and control conditions (no addition of any primary substrate). 10 Fig. S5. Biotransformation pathway of iopromide and peak area ratios referred to the internal standard (d10-CBZ) of iopromide (in the left axis), and its metabolites TP805A and TP805B, TP819, TP817A (in the right axis), determined by LC/MS/MS for the control assay (no primary substrate) with nitrifying sludge (AS2). 11 Fig. S6. Fate of persistent PhACs (< 50 µg/g VSS·d and removal < 5% after 1 d) in both control assays (no additional substrate) performed with AS1 and AS2. The fate under heterotrophic conditions (acetate supply) and nitrifying conditions (ammonium supply) are also shown. 0.0 0.2 0.4 0.6 0.8 1.0 1.2 0 1 2 3 4 5 C/C0 Time (d) Diclofenac 0.0 0.2 0.4 0.6 0.8 1.0 1.2 0 1 2 3 4 5 C/C0 Time (d) Carbamazepine 0.0 0.2 0.4 0.6 0.8 1.0 1.2 0 1 2 3 4 5 C/C0 Time (d) Diclofenac 0.0 0.2 0.4 0.6 0.8 1.0 1.2 0 1 2 3 4 5 C/C0 Time (d) Trimethoprim 0.0 0.2 0.4 0.6 0.8 1.0 1.2 0 1 2 3 4 5 C/C0 Time (d) Trimethoprim 0.0 0.2 0.4 0.6 0.8 1.0 1.2 0 1 2 3 4 5 C/C0 Time (d) Carbamazepine Acetate supply Ammonium supply Control Heterotrophic sludge (AS1) Nitrifying sludge (AS2) 12 References Abegglen, C., Joss, A., McArdell, C.S., Fink, G., Schlüsener, M.P., Ternes, T.A., Siegrist, H., 2009. The fate of selected micropollutants in a single-house MBR. Water Research 43 (7), 2036-2046. Göbel, A., Thomsen, A., McArdell., C.S., Joss, A., Giger, W., 2005. Occurrence and sorption behavior of sulfonamides, macrolides and trimethoprim in activated sludge treatment. 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