The organic loading rate affects organic micropollutants’ cometabolic biotransformation kinetics under heterotrophic conditions in activated sludge

The organic loading rate affects organic micropollutants’ cometabolic biotransformation kinetics under heterotrophic conditions in activated sludge David M. Kennes-Veiga, Lorena Gonzalez-Gil, Marta Carballa, Juan M. Lema Accepted Manuscript How to cite: Water Research, 189 (2021), 116587 https://doi.org/10.1016/j.watres.2020.116587 Copyright information: © 2020 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/) HIGHLIGHTS 1. OMPs cometabolic biotransformation was proved with aerobic heterotrophs. 2. OMPs biotransformation yield did not correlate with the heterotrophic activity. 3. kbiol increased with the heterotrophic activity for most compounds. 4. The primary carbon source and the OMPs followed a simultaneous removal. *Highlights (for review) GRAPHICAL ABSTRACT Graphical Abstract 1 The organic loading rate affects organic micropollutants’ cometabolic 1 biotransformation kinetics under heterotrophic conditions in activated 2 sludge 3 David M. Kennes-Veiga*, §, Lorena Gónzalez-Gil†, Marta Carballa§ and Juan M. Lema§ 4 § CRETUS Institute, Department of Chemical Engineering, Universidade de Santiago de5 Compostela, 15782 Santiago de Compostela, Galicia, Spain 6 † Defence University Centre, Spanish Naval Academy, Plaza de España, 36920, Marín, Spain 7 * Corresponding author. Tel.: +34 881816784. E-mail address: david.k[email protected]8 9 ABSTRACT 10 Several studies have shown that organic micropollutants (OMPs) are biotransformed 11 cometabolically in activated sludge systems. However, the individual role of 12 heterotrophs in the microbial consortium is still not clear, i.e., there is still a gap 13 regarding the influence of the heterotrophic activity on the cometabolic 14 biotransformation kinetics and yield of the OMPs. Aiming to answer these questions, 15 experiments with increasing primary substrate concentrations were performed under 16 aerobic heterotrophic conditions in a continuous stirred tank reactor operated at several 17 organic loading rates (OLR) with fixed hydraulic retention time. Moreover, the 18 individual kinetic parameters were determined in batch assays with different initial 19 substrate concentrations using the sludges from the continuous reactor. A set of 15 20 OMPs displaying a variety of physicochemical properties were spiked to the feeding in 21 the ng L-1 - µg L-1 range. Results reveal that the biodegradation of the primary carbon22 source and the biotransformation of the OMPs occur simultaneously, in clear evidence 23 of cometabolic behaviour. Moreover, we conclude that the OMPs biotransformation 24 kinetic constant (kbiol) shows a linear dependence with the OLR of the primary substrate 25 for most of the compounds studied, suggesting that the heterotrophic activity seriously 26 affects the OMPs biotransformation kinetics. However, under typical activated sludge 27 systems operating conditions (hydraulic retention times above 8 h), their 28 biotransformation yield would not to be significantly affected. 29 Keywords: biotransformation kinetic constant; heterotrophs; pharmaceuticals; 30 wastewater treatment plant; yield 31 2 1. INTRODUCTION32 Organic micropollutants (OMPs) including industrial chemicals, pharmaceuticals, 33 personal care products, pesticides and hormones are released daily and carried in 34 residual waters from households, hospitals, industries and agriculture before they reach 35 wastewater treatment plants (WWTPs) (Luo et al., 2014). WWTPs are only capable of 36 removing them to a certain extent, depending on the compound and the process applied, 37 and have become the main source of OMPs release to the environment (Kasprzyk-38 Hordern et al., 2009). 39 Since OMPs are present in WWTPs at very low concentrations, in the ng L-1 and g L-1 40 range, they are not capable of supporting microbial growth and, therefore, a primary 41 substrate is required to induce the enzymatic action that allows OMPs biotransformation 42 through cometabolism (Fischer and Majewsky, 2014). As a result, OMPs are 43 biotransformed within the metabolic pathways of the microbial populations present in 44 WWTPs (Alvarino et al., 2018a; Müller et al., 2013). The most common biological 45 treatment in WWTPs is based on an activated sludge system, which typically involves 46 heterotrophic and autotrophic nitrifying activities (Gernaey and Sin, 2013). The 47 combination of nitrifiers and heterotrophs provides a broader range of enzymatic 48 activities, helping to remove a wider variety of OMPs and to a higher extent. For 49 instance, Fernandez-Fontaina et al. (2016) reported that the presence of aerobic 50 heterotrophs enhanced the removal of sulfamethoxazole in activated sludge, and Men et 51 al. (2016) determined that when the nitrifying population was inhibited the 52 biotransformation of bromoxynil was considerably reduced. 53 The action of heterotrophic bacteria is fundamental in most WWTPs since they are 54 responsible for the biological removal of organic matter (Majewsky et al., 2011). In 55 fact, some facilities are designed only with this purpose and the main microbial activity 56 3 they present is heterotrophic (Metcalf & Eddy, 2014). Moreover, the importance of 57 heterotrophs is gaining interest in the novel conceptions of WWTPs, where the A-58 stages, working at high organic loading rates (OLR) and short solid retention times 59 (SRT), operate basically under heterotrophic conditions (Jimenez et al., 2015). 60 Heterotrophs are fast growers, have short doubling times (K. Kim et al., 2020) and, 61 compared with autotrophs, possess a more efficient metabolism and a higher diversity 62 of organisms (Holtmann and Sell, 2002). Besides, Maeng et al. (2013) and Majewsky et 63 al. (2010) have indicated that heterotrophic bacteria may be involved in the removal 64 yield and degradation rates of OMPs when primary substrates are present. The positive 65 effect of the heterotrophic metabolism on the biotransformation of multiple OMPs has 66 been previously reported, as for diclofenac (Nguyen et al., 2019), 17-ethinylestradiol 67 (Larcher and Yargeau, 2013) and ibuprofen (Alvarino et al., 2018a), among others. 68 Furthermore, Fernandez-Fontaina et al. (2016) even showed that heterotrophs can be 69 more favourable than autotrophs for biotransformation, as in the case of 70 sulfamethoxazole. Nonetheless, due to the complexity of the heterotrophic metabolism, 71 the contribution they make to the effectiveness and the biotransformation kinetics of the 72 OMPs in WWTPs is still not clear. 73 To characterize OMPs biotransformation, a pseudo-first order kinetic model is 74 commonly assumed and the resulting biotransformation kinetic constant (kbiol) largely 75 depends on the physicochemical properties of each particular pollutant (Lema and 76 Suarez, 2017). In fact, Falås et al. (2016) concluded that kbiol varies more depending on 77 the nature of the OMPs than on the experimental conditions. However, kbiol, as a kinetic 78 constant, is also affected by the reactor operating conditions (such as temperature, pH 79 and oxidation reduction potential), the presence and availability of co-substrates and the 80 biochemical versatility of the sludge, as several studies demonstrate (Barceló, 2012; 81 4 Petrovic et al., 2013). For instance, Alvarino et al. (2016) reported kbiol values of 0.09 82 and 0.05 L g VSS-1 d-1 for sulfamethoxazole under aerobic heterotrophic and 83 autotrophic denitrifying conditions, respectively; Gulde et al. (2014) reported kbiol 84 increments of one order of magnitude for atenolol at pH 8 compared to pH 6 under 85 activated sludge conditions and Li et al. (2005) reported that 17-estradiol increased its 86 kbiol value from 1.8 to 3.3 L g VSS-1 h-1 when increasing the temperature from 20 to 87 35ºC in batch experiments with activated sludge. Nonetheless, conversely, there are so 88 far no conclusive studies regarding the influence of the OLR and the heterotrophic 89 activity on kbiol. 90 The role of the OLR in the removal of the OMPs is, in fact, a topic of current and 91 general interest in biological systems, but results still do not show a clear trend. For 92 instance, Kora et al. (2020) showed that increasing the OLR improved the removal of 3 93 out of 5 OMPs spiked in a methanogenic-aerobic moving bed biofilm reactor and 94 Moya-Llamas et al. (2018) found better biodegradations for 6 OMPs at higher OLRs in 95 a UASB reactor coupled to a MBR. On the contrary, in a moving biofilm bed reactor 96 Abtahi et al. (2018) reported for some OMPs a maximum removal at the highest OLR 97 tested and for others at the lowest OLR and Carneiro et al. (2020) reported a negative 98 impact of increasing OLRs in the biodegradation of citalopram and sulfamethoxazole in 99 anaerobic fix bed biofilm reactors. Finally, differently to these studies, Gonzalez-Gil et 100 al. (2018b) found no correlation between variations in the OLR and the 101 biotransformation of most OMPs in methanogenic digesters. 102 The objective of the present study is to extend the knowledge behind OMPs 103 biotransformation processes under exclusively aerobic heterotrophic conditions. More 104 specifically, research is focussed on assessing the OMPs cometabolic biotransformation, 105 aiming to determine the relationship between the intensity of the heterotrophic activity 106 5 and the OMPs biotransformation rate, with a particular focus on evaluating the effect on 107 kbiol. The implications of understanding such linkage could be of great importance to 108 design biological systems able to maximize OMPs removal. To reach these goals, a 109 series of experiments in a continuous stirred tank reactor (CSTR) operated at different 110 OLRs were performed, as well as batch experiments set with different initial chemical 111 oxygen demand (COD) concentrations. 112 113 114 2. MATERIALS AND METHODS 115 2.1 Organic micropollutants 116 This study focused on 15 OMPs commonly present in WWTPs that display a wide 117 variety of chemical structures, physicochemical properties and applications (Table S3). 118 The compounds were: the antibiotics erythromycin (ERY), roxithromycin (ROX), 119 trimethoprim (TMP), sulfamethoxazole (SMX); the anti-inflammatories ibuprofen 120 (IBP), naproxen (NPX); the neuro drugs carbamazepine (CBZ), diazepam (DZP); the 121 musk fragrances celestolide (ADBI), galaxolide (HHCB), tonalide (AHTN); the biocide 122 triclosan (TCS) and the endocrine disruptors estrone (E1), 17-estradiol (E2), 17-123 ethinylestradiol (EE2). The OMPs were purchased from Sigma-Aldrich (Germany), 124 except for the musk fragrances, which were acquired from Ventos (Spain). Depending 125 on the substance, stock solutions were prepared in HPLC-grade acetone or methanol 126 and stored at -20C. 127 2.2 Aerobic Heterotrophic Reactor 128 A 5 L continuously stirred lab-scale reactor (Figure S1), connected to a 2 L settler, was 129 set at 25C and operated at four different OLRs (0.2, 0.4, 0.6 and 0.8 g COD L-1 d-1, that 130 6 resulted in 0.4, 0.6, 1.1 and 2.7 g COD g VSS-1 d-1), selected trying to cover typical 131 conditions of activated sludge WWTPs (Lema and Suarez, 2017; Metcalf & Eddy, 132 2014). Each experimental stage was maintained for one month and the OLR was 133 changed by varying the organic matter concentration in the feeding while keeping 134 constant the hydraulic retention time (HRT). The reactor was inoculated with sludge 135 from a conventional activated sludge reactor of a WWTP near Santiago de Compostela 136 (Spain). The WWTP was designed for 184000 population equivalents, receives an 137 influent COD ranging between 0.2 - 0.7 g L-1 and operates with approximate SRT and 138 HRT values of 10 d and 8 h, respectively. The feeding consisted of a synthetic mixture 139 of sodium acetate and acetic acid as primary carbon source (in concentrations that 140 ensured operation at neutral pH), ammonium chloride, potassium dihydrogen 141 phosphate, calcium chloride and magnesium sulfate (Table S1). Acetate was selected as 142 the carbon source for being an easily biodegradable substrate, optimal for microbial 143 growth, as well as for being directly metabolized through the Krebs cycle (Nelson and 144 Cox, 2017). Even though varying carbon sources lead to the expression of different 145 enzymatic activities and to varying enzymatic regulation levels, the mineralization of 146 organic matter typically requires the involvement of the Krebs cycle. Thus, the use of 147 acetate ensures that the enzymatic activities present in the bioreactor are also present in 148 WWTPs that deal with more complex carbon substrates. Moreover, other trace nutrients 149 were also added to promote the growth of aerobic heterotrophic microorganisms (Table 150 S2). The HRT was set to 1 d to provide sufficient time to the microorganisms to achieve 151 the maximum biotransformation they are capable of, at the same time as minimizing 152 possible changes in the microbial population caused by an HRT too long. Besides, to 153 avoid nitrification, allylthiourea solution was added in the feeding with a concentration 154 of 5 mg L-1 and the SRT was maintained below 8 d to minimize the presence of slow-155 13 F outlet flow rate (L d-1) 292 2.6 Statistical analysis 293 To determine if the yields and specific biotransformation rates of the OMPs were 294 statistically different at the tested primary substrate specific biodegradation rates, R 295 software 3.6.2. was used. The statistical tests were performed at a 5% significance level 296 (p < 0.05). 297 298 299 3. RESULTS AND DISCUSSION 300 3.1 Biotransformation yield 301 Fig. 1 shows the biotransformation yield, i.e. the biotransformation percentage, of the 302 selected OMPs at the 4 organic loading rates in the aerobic heterotrophic reactor. The 303 biotransformation extent varied considerably among OMPs, being their behaviour 304 characterized as (i) low biotransformation (below 20%) for CBZ and DZP, (ii) medium-305 low biotransformation (20-50%) for TMP, (iii) medium-high biotransformation (50-306 80%) for ERY and (iv) high biotransformation (over 80%) for ROX, SMX, IBP, NPX, 307 TCS, ADBI, HHCB, AHTN, E1, E2 and EE2. These results are consistent with 308 literature information (Alvarino et al., 2018b; Fernandez-Fontaina et al., 2016; Khunjar 309 et al., 2011). 310 Figure 1 311 It can be observed that the biotransformation yield of most OMPs remained constant 312 throughout the different OLRs (biotransformation differences below 10 percentage 313 14 points and not statistically significant; p > 0.05 ). Only one value for ERY (experiment 314 with 1.1 g COD g VSS-1 d-1) and another for IBP (experiment with 2.7 g COD g VSS-1 315 d-1), significantly (p < 0.05) deviated (between 15-20 percentage points) to the values of 316 the other three conditions. These events for ERY and IBP cannot be attributed to a 317 specific trend or behaviour. Therefore, overall, the results of Fig. 1 indicate that 318 increasing the aerobic heterotrophic cometabolism did not affect the OMPs 319 biotransformation yield under the conditions tested. Several hypotheses could explain 320 this behaviour. The first one is that the enzymes involved in the OMPs 321 biotransformation might not be increasingly stimulated at higher OLRs. According to 322 Stadman (1970), in catabolic pathways, enzymes can be classified as “constitutive” 323 enzymes, whose concentration is independent of the presence of their substrates, and 324 “inducible” enzymes, that are produced when their immediate substrates or suitable 325 derivatives are present. If the enzymes involved in the biotransformation of the OMPs 326 belong to the first class and were not increasingly stimulated, the yield could have 327 remained stable even at higher OLRs. A second hypothesis is that the maximum 328 cometabolic rate towards OMPs is already reached at the lowest OLR and, therefore, 329 increasing the degradation rate of the primary substrate (acetate) does not have an effect 330 on the OMPs. In this regard, it is commonly assumed that the oxidation rate of a non-331 growth substrate (OMPs) should always be linked to the oxidation rate of a growth 332 substrate (acetate) proportionally; however, some studies have shown that this is not 333 necessarily always the case (Criddle, 1993; M. H. Kim et al., 2020). Thirdly, the OMPs 334 may have already achieved their biotransformation limits due to thermodynamic 335 constraints. This event could be caused by the reversibility of the biological reactions, 336 leading to a chemical equilibrium between the parent compound and the transformation 337 product, as it has been previously suggested (Gonzalez-Gil et al., 2019). Lastly, the 338 15 primary metabolism may increase the specific biotransformation rate, but the HRT may 339 have been high enough to hide this effect, showing the same biotransformation yield in 340 all cases. In common biological treatments, as in activated sludge, the optimal HRT for 341 OMPs removal is 24 h or longer (Boonnorat et al., 2019). Nevertheless, it has been 342 proven that lower times may be sufficient to achieve the maximum biotransformation 343 extent for several compounds (Boonnorat et al., 2019; Ejhed et al., 2018). Overall, the 344 results shown in Fig. 1 are insufficient to determine which of these four hypotheses is 345 more likely, being necessary to evaluate the behavior of the biotransformation rate. 346 3.2 Specific biotransformation rate 347 In Fig. 2, it can be observed the direct relationship between the OMPs biotransformation 348 rate and the specific sludge activity: the higher the specific biodegradation rate of the 349 growth substrate, the higher the OMPs specific biotransformation rate (Eq. 9). The 350 dependence of the biotransformation rate of the secondary substrates (OMPs) with the 351 biodegradation of the primary substrate (acetate) is a clear prove of cometabolism 352 (Lema and Suarez, 2017). When the concentration of the primary substrate is increased 353 (i.e., a higher OLR is applied), a higher microbial activity of the biomass is achieved. 354 Hence, if an increment of the OMPs biotransformation rate also occurs, it indicates that 355 they are been cometabolized by the same enzymes involved in the metabolism of the 356 growth substrate. In Fig. 2, the differences in the specific biotransformation rates were 357 statistically significant (p < 0.05) for all experimental conditions and all OMPs tested. 358 Figure 2 (A, B and C) 359 Despite all OMPs following a cometabolic biotransformation trend, there are some 360 differences between the compounds, which do not show a homogeneous impact of the 361 OLRs in their biotransformation rates. For instance, while the biotransformation rate of 362 16 TCS or SMX only slightly increases between the conditions of 0.4 g COD g VSS-1 and 363 0.6 g COD g VSS-1, other compounds such as ADBI, HHCB and AHTN are much more 364 affected. The explanation for this variability could be related to the enzymatic 365 biotransformations. It is very likely that the OMPs are biotransformed by different 366 enzymes present in the heterotrophic metabolism and that their affinity for such 367 enzymes varies, leading to changes in their cometabolic biotransformation rates. In fact, 368 according to Kim et al. (2020), the cometabolic biotransformation is influenced by the 369 ratio of the initial growth to non-growth substrate concentration and the specificity 370 constant (the kinetic efficiency, that measures how efficiently an enzyme converts 371 substrates into products) ratio of the growth and non-growth substrates, which could be 372 different for each compound. 373 Overall, the specific OMPs biotransformation rates at 2.7 g COD g VSS-1 d-1 were 374 around 2.5- and 4-times fold the values obtained in the experiment performed at 0.4 g 375 COD g VSS-1 d-1. Nevertheless, it is noticeable that in comparison to the other 376 compounds, ERY, TMP, E1, E2 and EE2 (Fig. 2) showed reduced rate increments 377 between the experiments performed at 1.1 and 2.7 g COD g VSS-1 d-1 (although still 378 significantly different (p < 0.05)). These lower changes at the higher OLRs could 379 indicate that those compounds are close to reaching a maximum cometabolic 380 biotransformation rate (Gonzalez-Gil et al., 2018), likely determined by some 381 thermodynamic constraints, as suggested in the third hypothesis of section 3.1. In any 382 case, Fig. 2 shows that neither the first hypothesis (enzymatic stimulation was not 383 occurring at higher OLRs) nor the second (the maximum cometabolic rate had been 384 achieved at the lowest OLR) were taking place. Therefore, the best explanation for the 385 results of Fig. 1 is that, although the biotransformation rates varied with the OLR, the 386 HRT was high enough to hinder an effect on the biotransformation yield. 387 17 Previous studies have also proven the cometabolism of OMPs in different 388 environments. For example, Fernandez-Fontaina et al. (2012) reported a cometabolic 389 effect in the biotransformation of IBP, ERY, ROX and AHTN, among others, in 390 nitrifying reactors, likely due to the action of ammonia monooxygenase. Similarly, 391 Majewsky et al. (2011) determined that the heterotrophic active fraction was correlated 392 with the removal extent of pharmaceuticals such as DCF and SMX. Interestingly, under 393 anaerobic conditions, González-Gil et al. (2018b) reached a methanogenic activity high 394 enough not to show a relationship with the biotransformation rate of most OMPs, 395 contrary to previous anaerobic studies with lower activities of the primary substrate 396 (Alvarino et al., 2014). 397 The cometabolic effect proved in this study might imply that reactors operating at 398 higher OLRs could have an improved performance. Higher OLRs are often related to an 399 increased microbial activity and to a larger expression or activity of the enzymes 400 involved in the metabolism of the primary substrates, leading to a higher catalytic 401 activity in the reactor which could enhance the cometabolic biotransformation of the 402 OMPs. Therefore, high load WWTPs are likely to have a better performance in OMPs 403 biotransformation by increasing their yield or, if a limitation has been reached (as in 404 Fig.1), by being able to achieve in a shorter amount of time the same biodegradation 405 yield than low load WWTPs, allowing operations at shorter HRTs. 406 3.3 Influence of heterotrophic activity on the OMPs biotransformation constant 407 In previous sections, it was shown that a higher heterotrophic activity leads to a higher 408 specific OMPs biotransformation rate, indicating that heterotrophs are key on rbiol. 409 Based on Eq. 6, it seems that the influence of the heterotrophic activity on rbiol occurs 410 through changes on kbiol. Thus, batch tests were conducted to determine the correlation 411 18 between the microbial activity and kbiol, as well as between the removal of the primary 412 substrate and the pollutants. 413 Studying the interaction between the OMPs and the growth substrates can help to 414 understand their dynamics and control the fate of the pollutants by properly managing 415 the primary substrates. As shown in Fig. 3, the biotransformation of the OMPs 416 (exemplified for ROX, SMX, NPX and HHCB) occurs simultaneously to the 417 degradation of the primary carbon source (acetate). This finding agrees with the 418 common cometabolic biotransformation theory, where the OMPs, induced by the 419 presence of the primary substrate, enter its metabolic pathway and undergo 420 simultaneous biodegradation (King et al., 1997). OMPs do not yield enough energy to 421 support microbial growth and their biotransformation occurs fortuitously when their 422 chemical structure is modified thanks to the enzymatic action that degrades the primary 423 substrate. Similarly to the findings shown in Fig. 3, Aeming et al. (2019) determined 424 that the removal of 4 endocrine disruptors (fluoranthene, benzo(b)fluoranthene, 425 benzo(a)pyrene and NP) had a synchronal fate to the organic matter during anaerobic 426 digestion and composting. According to the results shown in Fig. 2 and Fig. 3, it is 427 proved that the main driver during the heterotrophic biotransformation of OMPs is the 428 presence of the growth substrate due to cometabolism. 429 Figure 3 430 As previously indicated, the kbiol of a particular compound depends on its 431 physicochemical properties and in the environmental conditions (pH, temperature and 432 oxidation reduction potential, among others). Interestingly, as shown in Fig. 4, we found 433 out that the specific activity of the sludge, caused by the primary substrate, also affects 434 the value of kbiol. A positive correlation (with an R-square value between 0.80 and 0.96) 435 is observed between kbiol and the maximum COD specific activity for all non-436 19 recalcitrant OMPs, with the exceptions of HHCB and ADBI. This proves that rbiol is 437 influenced by the microbial activity through the variation of kbiol. Since it was not seen a 438 limitation in the increasing tendency of kbiol, which maintained the linearity throughout 439 the assays, it can be speculated that higher kbiol values could be obtained at even higher 440 specific COD activities. Nonetheless, we do not have enough data to prove this theory 441 and experiments at higher heterotrophic activities would be required for confirmation. 442 It is worth comparing the results from Fig. 2 and Fig. 4, which showed some 443 differences. For instance, ERY showed almost 4-fold increases in its specific 444 biotransformation rate (Fig. 2) while its kbiol values only showed 2-fold increments (Fig. 445 4). These discrepancies were also observed in other compounds such as E1 and ROX. 446 One explanation for such behaviour could be that media conditions and especially 447 process conditions, such as operating in batch mode rather than in a continuous manner, 448 could lead to a different intensity in the metabolic responses, in agreement with the 449 results from Park et al. (2018). Besides, microorganisms may have followed different 450 enzymatic routes in the batch and reactor experiments to degrade the substrates due to 451 variations in the initial feeding concentrations, in consonance with previous studies 452 showing that the concentration of the metabolites plays a key role in the pathway 453 selection and metabolic flux (Wegner et al., 2015). Despite these differences, overall 454 both continuous and batch experiments provide consistent and complementary 455 conclusions. 456 Figure 4 (A and B) 457 Throughout the experiments, the influence of the heterotrophic activity on kbiol varied 458 among the OMPs. For instance, in the case of the hormones, while E1 increased its kbiol 459 almost 20-fold, E2 only increased it 2-fold and in the case of the fragrances, while 460 20 AHTN duplicated its kbiol, ADBI and HHCB did not show any variation. These findings 461 show that the influence of the heterotrophic activity is substance-specific. 462 The reported results show that the biotransformation of a particular OMP would depend 463 on the characteristics of the microbial population and their specific performance. From 464 our results, and considering the findings of Majewsky et al. (2010) and Gonzalez-Gil et 465 al. (2018b), it can be concluded that at low microbial activities, the kbiol of a compound 466 does not vary, at intermediate activities the kbiol increases in a linear manner, and 467 finally, when the microbial activity is sufficiently high, it reaches a plateau and kbiol 468 remains constant. Our findings suggest that, for the range of heterotrophic activity 469 studied, the majority of the selected OMPs were in the region where kbiol increases 470 linearly. The exceptions of HHCB and ADBI could indicate that they would have 471 required lower or higher microbial activities, proving that kbiol is influenced both by the 472 microbial activity and the nature of the compound. 473 To better understand the influence of the microbial activity on the biotransformation of 474 the OMPs, Table 1 shows an estimation of the biotransformation rates that could be 475 achieved for ROX, NPX, SMX and HHCB (the remaining compounds can be found in 476 Table S6) in a real activated sludge system designed to only remove organic matter, as 477 well as the HRT that these OMPs would require for complete biotransformation 478 according to the experimental kbiol values (Fig. 4). For instance, in the case of NPX, 479 assuming an inlet concentration of 10 g L-1, a kbiol equivalent to 0.7 L g VSS-1 d-1 480 would provide a biotransformation rate of 14 g NPX L-1 d-1 and an HRT of 17 h would 481 be required. On the other hand, a kbiol of 3.8 L g VSS-1 d-1 would lead to a 482 biotransformation rate of 76 g NPX L-1 d-1 and an HRT of 3 h could be sufficient for 483 total NPX removal. The results indicate that higher heterotrophic activities could 484 considerably reduce HRT requirements thanks to improved OMPs specific 485 21 biotransformation rates. Moreover, except for DZP and CBZ, which are recalcitrant 486 under heterotrophic conditions, all the OMPs would be almost completely removed 487 after 8 h (Tables 1 and S6). Accordingly, promoting the heterotrophic activity with 488 higher OLRs would not have significant effects on the biotransformation yield at typical 489 HRTs of activated sludge systems, supporting the findings of Fig. 1. 490 22 Table 1. Biotransformation rate and HRT required for full biotransformation of ROX, NPX, SMX and HHCB in a real WWTP based on the kbiol 491 values obtained in the batch assays. The XVSS value used is a typical solids concentration in WWTPs (Metcalf & Eddy, 2014), and the Cw 492 value is in the range of typical influent OMPs concentrations in WWTPs (Luo et al., 2014; Petrie et al., 2014). 493 OMPs Specific biotr. rate (g COD g VSS-1 d-1) kbiol (L g VSS-1 d-1) XVSS (g L-1) CW ( OMP L-1) Biotransformation rate ( g OMP L-1 d-1) HRT required for 100% biotransformation (h) ROX 0.4 1.6 2 10 32 7.6 0.6 2.4 49 4.9 1.1 3.4 68 3.5 2.7 4.0 81 3.0 NPX 0.4 0.7 2 10 14 17.1 0.6 2.8 56 4.3 1.1 3.4 68 3.5 2.7 3.8 76 3.2 SMX 0.4 1.0 2 10 20 12.0 0.6 2.0 40 6.0 1.1 2.6 52 4.6 2.7 2.9 58 4.1 HHCB 0.4 5.0 2 10 100 2.4 0.6 3.9 78 3.1 1.1 6.7 134 1.8 2.7 5.2 104 2.3 29 5th ed. McGraw-Hill, New York (USA). 672 Moya-Llamas, M.J., Trapote, A., Prats, D., 2018. Removal of micropollutants from 673 urban wastewater using a UASB reactor coupled to a MBR at different organic 674 loading rates. 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How metabolites modulate 729 metabolic flux. Curr. Opin. Biotechnol. 34, 16–22. 730 https://doi.org/10.1016/j.copbio.2014.11.008 731 732 Fig. 1. OMPs biotransformation yield achieved in the aerobic heterotrophic reactor operated with fixed HRT (1 day) at 4 different specific organic loading rates of the primary substrate. 0 10 20 30 40 50 60 70 80 90 100 ERY ROX SMX TMP IBP NPX TCS ADBI HHCBAHTN E1 E2 EE2 CBZ DZP Biotransformation (%) 0.4 g COD/g VSS·d 0.6 g COD/g VSS·d 1.1 g COD/g VSS·d 2.7 g COD/g VSS·d Fig_1 A) B) 0.0 0.5 1.0 1.5 2.0 E1 E2 EE2 Biotransformation rate ( µ g OMP biotransformed /g VSS·d) 0.4 g COD/g VSS·d 0.6 g COD/g VSS·d 1.1 g COD/g VSS·d 2.7 g COD/g VSS·d 0 10 20 30 40 50 60 ADBI HHCB AHTN Biotransformation rate ( µ g OMP biotransformed /g VSS·d) 0.4 g COD/g VSS·d 0.6 g COD/g VSS·d 1.1 g COD/g VSS·d 2.7 g COD/g VSS·d Fig_2 C) Fig. 2. Specific OMPs biotransformation rate (µg OMP g VSS-1 d-1) at 4 specific biodegradation rates of the primary substrate (g COD g VSS-1 d-1). A) OMPs fed at 1 µg L-1 d- 1. B) OMPs fed at 40 µg L-1 d-1. C) OMPs fed at 10 µg L-1 d-1. 0 5 10 15 20 25 30 ERY ROX SMX TMP IBP NPX TCS Biotransformation rate ( µ g OMP biotransformed /g VSS·d) 0.4 g COD/g VSS·d 0.6 g COD/g VSS·d 1.1 g COD/g VSS·d 2.7 g COD/g VSS·d Fig. 3. Representation of the biotransformation trend followed by ROX, SMX, NPX and HHCB for the experimental batch with [COD]0 = 0.4 g L-1. Similar results were obtained for the rest of OMPs and COD evaluated (data not shown). 0.00 0.25 0.50 0.75 1.00 012 24 36 48 C/C0 Time (h) NPX ROX SMX HHCB [COD] = 0.4 g/L Fig_3 A) kbiol (L/g VSS·d) Maximum specific activity (g COD/g VSS·d) R² = 0.96 0 20 40 60 0 1 2 3 4 5 E1 R² = 0.87 0 20 40 60 0 1 2 3 4 5 E2 R² = 0.83 0 1 2 3 0 1 2 3 4 5 EE2 R² = 0.92 0 1 2 3 012345 ERY R² = 0.89 0 2 4 6 0 1 2 3 4 5 NPX R² = 0.97 0 2 4 6 0 1 2 3 4 5 ROX R² = 0.84 0 2 4 6 0 1 2 3 4 5 IBP R² = 0.91 0 2 4 6 0 1 2 3 4 5 TCS R² = 0.94 0 2 4 0 1 2 3 4 5 SMX R² = 0.8 0 5 10 0 1 2 3 4 5 AHTN Fig_4 B) kbiol (L/g VSS·d) Maximum specific activity (g COD/g VSS·d) *The kbiol of TMP could not be determined. Fig. 4. OMPs biotransformation kinetic constant (kbiol) vs the maximum COD specific activity achieved in each batch assay. A) OMPs that showed a positive correlation. B) OMPs that showed a negative correlation. In each graph is stated the R squared value (R2). R² = 0.06 0 5 10 15 0 1 2 3 4 5 HHCB R² = 0.02 0 5 10 15 0 1 2 3 4 5 ADBI R² = 0.28 0 1 2 0 1 2 3 4 5 CBZ R² = 0.45 0 1 2 0 1 2 3 4 5 DZP