Putative Role of Flavobacterium, Dokdonella and Methylophilus Strains in Paracetamol Biodegradation

Paracetamol, the most widely and globally used analgesic and antipyretic, is easily accumulated in aquatic environments. In the present study, the biodegradation of paracetamol in different media (one for general growth, one specific for sulfate reducing bacteria, a mineral salts medium and municipal wastewater) inoculated with two types of sludge (from anaerobic lagoon and from oxidation ditch) under different oxygenic conditions (anoxic; moderate oxygenation in open flasks and high oxygenation by aeration) was investigated. In addition, bacteria with relative abundances increasing simultaneously with paracetamol degradation, when this drug was the only carbon source, thus with a putative role in its degradation, were identified using 16S rRNA gene sequences. The results show that aerobic microorganisms had a major role in the degradation of paracetamol, with 50 mg/L totally removed from municipal wastewater after 2 days incubation with aeration, and that the metabolites 4-aminophenol and hydroquinone plus one compound not identified in this work were produced in the process. The identification of bacteria with a role in the degradation of paracetamol revealed a strain from genus Pseudomonas with the highest final relative abundance of 21.2%, confirming previous works reporting strains of this genus as paracetamol decomposers. Besides, genera Flavobacterium, Dokdonella and Methylophilus were also in evidence, with initial relative abundances of 1.66%, 1.48 and 0.00% (not detected) in the inoculum and 6.91%, 3.80 and 3.83% after incubation, respectively. Therefore, a putative role of these genera in paracetamol biodegradation is suggested for the first time. Graphical Abstract ᅟ ᅟ

Below are listed, one by one, the corrections made in the manuscript and the answers to the comments and questions posed. In the manuscript, new sentences and corrected sentences are written in green color.

Cordial regards Jorge Dias Carlier
Reviewer #1: General Comments: This work focused on biodegradation of paracetamol in wastewater and identifies three new genera with a putative role in paracetamol degradation based on their enrichment in cultures when that pharmaceutical was provided as the sole carbon source. The dominant phylotype identified in the cultures investigated was Pseudomonas, which had previously been shown to degrade paracetamol; however, the other phylotypes identified had not previously been linked with degradation and were shown here to likely play a role in biodegradation of paracetamol metabolites. The manuscript offers some novel findings on a relevant topic; however, I have some concerns. 1) A general concern relates to the lack of biomass measurement and biomass normalization between conditions. Before the authors conclude that biodegradation does not occur under certain conditions, they should demonstrate that biomass was not limiting, at least to the extent possible. For example, it would have been appropriate to at least document that initial biomass levels were the same across conditions. How biomass was normalized is not clear. Authors answer: It can be considered that a rough normalization was guaranteed in the beginning of all experiments because: (1) similar bacterial growth rates were observed in all inocula enrichments, (2) at the end of all enrichments the bacterial growth was at a roughly equivalent stage and (3) the inocula proportions were in all cases the same: 10% (v/v). The bacterial growth was evaluated by optical densities measurements in the beginning and at the end of the enrichment cultures (see also point 9 of specific comments below).
2) Second, it is not clear how replication was handled for DNA sequencing; thus, the reproducibility of key findings is not clear. Were the three phylotypes listed in the title enriched to the same degree over time in all biological replicates? Authors answer: This issue has been clarified. Corrections were made in the first sentence of section 2.7 to clarify that large-scale biodiversity analysis by DNA sequencing was performed along the experiment on samples from just one of the triplicated cultures. Moreover, the following sentences were added to section 2.7: -"As the three replicate cultures were inoculated exactly with the same microbial community (source: enriched culture from WWTP's oxidation ditch sludge; quantity: 10% (v/v)) and were maintained under exactly the same conditions (aeration: a shared air pump; temperature and light: flasks maintained side by side), it was assumed that studying the microbial dynamics through massive sequencing of 16S rRNA genes on just one of the cultures would provide enough robust evidences to identify taxa putatively involved in the degradation of paracetamol and/or its metabolites." Introduction 3. The Introduction should more clearly discuss the role of redox in paracetamol degradation. Are all the known pathways aerobic? What was the specific knowledge gap being addressed? Authors answer: done The following sentences were added to Introduction: -"Although paracetamol stability decreases in acidic or alkaline conditions due to be slowly degraded via a base-or acid-catalyzed hydrolysis of the amide bond into acetic acid and 4-aminophenol, stability studies in purified water at room temperature demonstrated that after incubation for more than one month this drug was completely stable due to the high energy needed to overcome the barrier for the cleavage of its amide bond (Karaman et al. 2016). In addition, being 4-aminophenol a photo and thermal sensitive compound, like other aromatic amines, the results obtained by Khan et al (2006) showed the occurrence of its autoxidation after 7 days but at low rates: 0.8%, 26.6%, 20% and 13% at 50, 100, 150 and 200 mg/L, respectively. In any case, the authors of both works concluded that biodegradation was more effective. Indeed, several studies have demonstrated that microorganisms play a major role in the environmental degradation of paracetamol and during the past decades research related to the biodegradation of this drug has allowed the identification and characterization of metabolic intermediates involved in the catabolic pathways of aerobic bacteria (Wu et al. 2012)." ... Minor corrections were made in the sentence of the Introduction starting by: -"In this regard, biodegradation may represent a low-cost effective solution (Wu et al. 2012), though..." ... The following sentence was added to the Introduction: -"Thus, to our knowledge all the microbial catabolic pathways known to be involved in paracetamol biodegradation were studied in a small number of aerobic organisms; therefore, it is possible that unidentified mechanisms with a role in paracetamol biodegradation exist in other organisms." ... 4. In the introduction, page 2, the statement the microorganisms degrade organic compounds is overly general and vague. Further, please also mention that some metabolites do not represent molecules that are particularly "simple". The issue of toxic metabolites should be addressed in the Introduction. Authors answer: done The following sentences were added to Introduction: -"When paracetamol is used in excess, it can cause liver failure and necrosis due to N-Acetyl-p-benzoquinone imine (NAPQI), a highly toxic paracetamol´s metabolite formed by enzymatic oxidation in the liver (Bessems and Vermeulen 2001). About 30% and 55% of administered paracetamol is excreted in urine as conjugates paracetamol sulfate and paracetamol glucuronide, respectively (Thomas 1993). Moreover, a glutathione conjugate (1,4-Michael adduct) of NAPQI, the corresponding cysteine conjugate and mercapturic acid breakdown products have also been found in urine after ingestion of paracetamol (Prescott 1980). NAPQI is known to be fairly unstable, however, in aqueous solution it readily hydrolyzes into 1,4-benzoquinone, which is another toxic metabolite (Dahlin and Nelson 1982;Snyder 2000;Bedner and Maccrehan 2006). On the other hand, 4-aminophenol (the hydrolytic product of paracetamol) has highly genotoxic and mutagenic effects (Majeska and Holden 1995;Yoshida et al. 1998). The potential toxic effects of paracetamol in aquatic systems, mediated by its reactive oxygen species, have been reported. For example, Antunes et al. (2013) studied the effects of paracetamol exposure on physiological traits of bivalves and the results showed a significant increase in all oxidative stress biomarkers, evidencing the onset of deleterious effects." ... The following sentences were removed from the Introduction: -"Microorganisms, particularly bacteria, have been demonstrated to play an important role in the biodegradation of organic compounds in WWTPs. Biodegradation can be partial or complete." ... Minor corrections were made in the sentence on the Introduction starting by: -"When biodegradation is complete (mineralization), the substances are converted to inorganic..." 5. How long were cultures enriched?
The following sentences were added to section 2.2: -"To enrich the inocula from the lagoon system, 2 g of sludge sediment were inoculated on each of two separate 250ml culture flasks containing 200 mL of TGM medium. In one flask 10 mL of paraffin-oil was added to prevent gas diffusion and the flask was sealed with a butyl rubber stopper and an aluminum crimp seal to avoid air intake in order to create anaerobic condition. In the other flask, cotton was used to serve as lid, thus allowing the maintenance of aerobic conditions. To enrich the inoculum from the oxidation ditch, 2 mL of water with sludge was inoculated in just one flask with 200 mL TGM medium with cotton as lid. The anaerobic flask was kept without shaking and the two aerobic flasks were placed in an orbital shaker at a speed of 150 rpm; all were maintained at room temperature and grown for 24 hours. The anaerobic enriched culture was used to inoculate all tests under anaerobic conditions. The aerobic enriched cultures from each type of sludge were used to inoculate the respective tests under moderate and highly aerobic conditions." ... Minor corrections were made in the remaining sentences of section 2.2.
Materials and Methods 6. Please provide more details and references on the liquid paraffin method used for promoting aerobic conditions. Authors answer: The use of liquid paraffin is to promote anaerobic conditions, not aerobic. The following sentence was added to section 2.4: "The liquid paraffin prevents gas diffusion through the medium surface and the flask sealed with a butyl rubber stopper and an aluminum crimp seal avoids air intake into the flask; thus the oxygen initially present is rapidly consumed and anaerobic conditions are created." 7. How did oxygen concentration vary between the "moderate" and "full" oxygenation conditions? What was the redox measurement? For moderate oxygenation cultures, were they stagnant? Or mixed? If mixed, how so? Authors answer: additional corrections were made in section 2.4 to address these issues. Minor corrections were made in the sentence of section 2.4 starting by: -"To maintain aerobic conditions with moderate oxygenation, the cultures were incubated in open batch flasks..." ... The following sentences were added to section 2.4: -"The averages and standard deviations of dissolved oxygen (DO) percent saturation in the various tests under aerobic conditions were calculated using measurements made in the respective replicate cultures with a portable CD650 meter (Eutech Instruments) after 24 hours of incubation since inoculation. For moderate oxygenation conditions in open flasks without aeration, the DO percent saturation was: 9.3±1.8% in the tests with TGM, 31.1±2.3% in the tests with MSM and 27.5±2.2% in the tests with MWW (24 hours after both first media were inoculated with the anaerobic lagoon sludge and the third medium inoculated with the oxidation ditch sludge). For high oxygenation conditions in aerated flaks, the DO percent saturation was: 94.8±1.6% in the tests with MSM and 95.5±1.3% in the tests with MWW (24 hours after both these media were inoculated with the oxidation ditch sludge)." 8. Please describe any pre-acclimation. Biodegradation rates are typically improved by pre-acclimating cultures. Authors answer: It was not performed any pre-acclimation with paracetamol. 9. How were biomass levels normalized between conditions? How was biomass measured? Authors answer: It can be considered that a rough normalization was guaranteed. The following sentences were added to section 2.2: -"In the inocula enrichments, initial and final optical densities were measured at 600 nm (OD600) as absorbance using a Hach-Lange spectrophotometer DR-2800 (Sköndal, Sweden). The initial OD600 values of enrichment media immediately after inoculation with the anaerobic lagoon and the oxidation ditch sludges were similar: 0.286 and 0.264, respectively. Thus, the also similar OD600 values measured after the 24 hours of incubation at room temperature in all enrichment conditions (anaerobic lagoon sludge/anaerobic growth = 0.838; anaerobic lagoon sludge/aerobic growth = 0.831; oxidation ditch sludge/aerobic growth = 0.833) roughly indicates similar bacterial growths." ...
-"The initial and the final OD600 values in the SRB inoculum enrichment were 0.165 and 0.657, respectively." 10. I'm unconvinced that the italicized subtitles are necessary. The first sentence of the DNA sequencing methods section is redundant with earlier text. Authors answer: We consider that the italicized subtitles are helpful for a more clear separation of all experiments and of all the several steps along the methodology; therefore we would like to maintain them. The first sentence of the DNA sequencing methods (section 2.7.2) was removed.
11. In 2.6.1, the medium list does not have to put each medium on a separate line.
Authors answer: Correction done as suggested.
12. Why were the tests only run for ~72 hours? This time might have been too short. Authors answer: We agree that it would have been better to run the tests for longer periods. The first set of tests, with the anaerobic lagoon sludge, was run just for 72 hours because it was thought that comparing paracetamol removals in the inoculated and the non inoculated cultures under the two conditions (anoxic vs low oxygenic) during that time could be enough to evaluate which major metabolic pathways contribute for the biodegradation of this drug in WWTPs: anaerobic, aerobic or both. The second set of tests, with the oxidation ditch sludge, was in fact maintained for more 3 than days (that is why in the bacterial dynamics study there is a sample in day 6). However, the samples collected on that day were not analyzed for the presence of paracetamol and respective degrading metabolites due to a prolonged failure in the HPLC equipment.
The following sentences were added to the beginning of section 3.2: -"The results obtained clearly indicate that the major routes for the biodegradation of this drug in WWTPs involve aerobic organisms; some already reported previously and others identified in this work for the first time, as discussed further below. However, some slow biodegradation seems to occur under anaerobic conditions, which was not more evident due to the short time of the assays." Results 13. OD600 is not a measure of bacterial "viability". You can have a high OD600 in killed cultures. I suggest changing the axis label for Figure. 2. Authors answer: The vertical axis label of Figure 2 is not OD600. In fact it is "Bacterial viability %", defined as the percentage of the optical density measured in a culture with a certain paracetamol concentration in relation to the optical density in the control culture without the drug. Corrections were made in section 2.3, in the sentence starting by: -"Based on the OD600 values, the percentage of bacterial viability..." ... Corrections were made in section 3.1, in the sentences starting by: -"The studies started by the determination of paracetamol IC50..." ...
-"Estimates were based on..." ... The following sentence was added to caption of Figure 2: -"Bacterial viability % is the optical density at 600 nm (OD600) measured in a culture with a certain drug concentration divided by the OD600 in the control culture without drug, multiplied by 100." 14. Do you have biomass data for the differing redox conditions? How is it known that biomass was not simply limiting at lower redox? Could low biomass have limited rates such that at anaerobic redox more time would have been required to observe statistically significant degradation? Authors answer: We agree that this issue is important.
The following sentences were added at the end of section 3.2.5: -"Though the biomass growth was not analyzed during the biodegradation tests, it was evaluated by optical density measurements at the beginning and at the end of the inocula enrichment in TGM (a rich medium for anaerobic and aerobic microorganisms) and during that period roughly similar growths were observed in the different redox conditions (as described in section 2.2). Taking that into account, together with the fact that roughly similar biomasses were inoculated in all tests, it can be assumed that at least for the tests with TGM the bacterial growth under anaerobic conditions was not a limiting factor. However, when MWW was used, the lower paracetamol degradation rates observed under low oxygenic conditions (27.5±2.2% DO) compared with those observed under high oxygenic conditions (95.0±1.3% DO) were probably the result of slower biomass growth rates in the former compared with the second. This can be considered expected due to the fact that in high oxygenic conditions the complex organic molecules present in MWW are faster oxidized and transformed into simpler carbon sources suitable for bacteria." 15. Which abiotic mechanisms account for the observed losses in un-inoculated controls?
Authors answer: this issue is now addressed.
The following sentences were added to section 3.2.5: -"One non biological mechanism that can account for the observed losses of paracetamol in all tested media is its oxidation by SO42-ions. It is known that compared with hydroxyl radical (•OH), SO42-is more selective for oxidation by electron-transfer reaction and more powerful for the decomposition of contaminants at neutral pH (Mezyk et al. 2011;Zhang et al. 2015). Indeed, when peroxymonosulfate (PMS) is activated by catalysts it decomposes generating SO42-, which degrades pollutants. Thus, several catalysts have been explored for PMS oxidation and some attracted great interest due to its remarkable separation and catalysis, such as spinel ferrites of MFe2O4 (M = Fe, Mn, Co, Ni, and Cu), (Tan et al. 2017). Another mechanism putatively accounting for the decrease of paracetamol detection in the experiments with TGM (including in the non inoculated controls) is the possible formation of protein-paracetamol complexes. It has been shown that paracetamol and serum proteins form complexes (e.g. Daneshgar et al. 2009) and the TGM has beef extracts in its composition." 16. Please include statistical analysis comparing inoculated and un-inoculated controls. E.g., in section 3.2.2, when it is stated that there is a 10% difference been inoculated and un-inoculated reactors, is this difference statistically significant? If this difference is not statistical, then modify the text that states a "small degradation caused by biological activity". Authors answer: Done.
The following sentence was added to the end of section 2.4: -"One-Way ANOVA (Single Factor) tests using Excel Data Analysis Tools were performed to evaluate if differences between the inoculated test cultures and the respective non inoculated negative controls were significant for 5% error (α = 0.05)." ... The following information was added to table 1: -"* = paracetamol removal is significantly different (one way ANOVA for 5% error) from the observed in the respective negative control." ... Corrections were made in section 3.2.1, in the sentences starting by: -"Even so, in all these cases and for both sampling days, the differences in paracetamol removal averages between the inoculated and non-inoculated tests were relatively low..." ...
-"In the inoculated TGM medium under aerobic conditions with moderate oxygenation..." ... The following sentence was removed from section 3.2.1: -"The discrepancy between these aerobic tests using TGM and the anaerobic tests with the same medium discussed above is that in aerobic conditions the removal of paracetamol observed after 72 h is not higher than it was after 24 h (Table 1) 17. It would be helpful if the subtitles also indicated the redox/electron acceptor tested. Authors answer: To avoid having long subtitles this information was added in the text.
The following sentence was added to section 3.2.1: -"The main electron acceptor in TGM is oxygen in the tests under aerobic conditions, while oxidized forms of inorganics such as nitrate, sulphate, iron (III) and manganese (IV), as well as amino acids and glucose are the potential electron acceptors in the tests under anaerobic conditions." ... The following sentence was added to section 3.2.2: -"This medium is optimized to cultivate SRB under anaerobic conditions, thus the main electron acceptor in its composition is sulphate." ... The following sentence was added to section 3.2.3: -"This medium, exclusively composed by inorganic compounds, was used to test paracetamol biodegradation when this drug was the only carbon source available. The putative main electron acceptors of MSM are oxygen, in the tests under aerobic conditions, and nitrate sulphate and manganese (IV), in the tests under anaerobic conditions." ... The following sentence was added to section 3.2.4: -"It was tested just under aerobic conditions, in which the main electron accepter is oxygen." 18. The last sentence of section 3.2.2 is entirely too long and should be broken up for readability.
Authors answer: Done. The last sentence of section 3.2.2 was substituted by this paragraph: -"These to some extent higher removals of paracetamol observed in the tests inoculated with the SRB enriched consortium compared to the respective non inoculated negative controls may indicate the existence of mechanisms associated with biological activity contributing to paracetamol removal. However, it remains unknown whether paracetamol was degraded via metabolic pathways, or if it reacted with compounds released by the active microbial communities. Despite this, it seems that the SRB present in this consortium are not directly involved in the removal because it occurred at the same extent both in medium with lactate (with high SRB activity) and without lactate (with low SRB activity). Thus, probably other microorganisms than SRB, eventually less abundant in the inoculum, may have been directly or indirectly responsible for the observed paracetamol removal." 19. Table 1 indicates "No sludge or autoclaved sludge", but these two options are not equivalent. Table 1 should indicate which was which.  The following information was added to table 1: -"c = negative controls carried out with autoclaved sludge." 20. Section 3.3, bottom of page 13: The drug is not "catalyzed". Reactions are catalyzed. Rephrase.
Authors answer: Done. The word "catalyzed" was substituted by the word "metabolized" and corrections were made in the respective sentence of section 3.3, which now stands like this: -"The occurrence of 4-aminophenol in all inoculated cultures and not in the negative controls (without sludge as inoculum) supports the major biodegradation pathway of paracetamol proposed for microbes by other authors: this drug is metabolized to produce 4-aminophenol, which is converted to hydroquinone through replacement of the amino group by a hydroxyl group, being then this aromatic compound the precursor of several carboxylic acids (2-hexenoic acid, succinic acid, malonic acid, oxalic acid and finally formic acid) (Hu et al. 2013;Wu et al. 2012;Zhang et al. 2013)." 21. Table 2 should indicate units.
Authors answer: Done.
The following information was added to table 1: -"OTU abundances (%)" 22. Figure 4 would be more useful if the peaks of interest were labeled somehow. This figure could optionally be moved to supplemental given its lack of complexity. Authors answer: The peaks of interest in figure 4 were labeled and we would like to maintain this figure as figure 4 in the paper.
23. How were replicated cultures handled during sequencing? Was sequencing conducted for all replicates? Was the data pooled? Replication is not displayed in Table 2. Was the identification of the phylotypes listed in the title reproducible across replicates? This is a critical point to address. Authors answer: Sequencing was conducted just for one of the three replicates. No samples, neither sequencing data was pooled. This point is now addressed.
The following sentences were added to section 3.5: -"The three replicate cultures of this test revealed identical behaviors for the concentrations of the analyzed compounds along the experiment. Thus, it was considered that the microbial dynamics studied through massive sequencing of 16S rRNA genes present in just one of the replicates along the incubation time would provide enough robust evidences to identify taxa putatively involved in the degradation of paracetamol and/or its metabolites." 24. Was sequencing conducted to separately measure Bacteria and Archaea? Or just Bacteria? Authors answer: Sequencing and respective bioinformatic processing was focused on bacteria.
The following sentence was added to section 3.5: -"The study was focused on bacteria using primers for the 16S rRNA gene region V1-3." 25. Dynamic trends in microbial community composition would best be visualized in a histogram rather than a table. Better yet, replicates could be displayed via principle component analysis.
Authors answer: We have created a histogram displaying the dynamic trends in microbial community composition for the 25 most abundant bacteria; however, we consider that it thus not improves the visualization and interpretation of results. Therefore, we would prefer to maintain Table 2.
Since sequencing was carried out in just one of the three replicates, we didn't try to display the results via principle component analysis.

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Highlights
-The IC50 of paracetamol for the growth of bacteria from municipal WWTP sludge was estimated.
-Biodegradation of paracetamol was tested in different media and oxygenic conditions.
-Flavobacterium, Dokdonella and Methylophilus are for first time associated to paracetamol degradation.
-The putative role of these genera in paracetamol degradation is discussed.

19
Although paracetamol stability decreases in acidic or alkaline conditions due to be slowly degraded via a 20 base-or acid-catalyzed hydrolysis of the amide bond into acetic acid and 4-aminophenol, stability studies in 21 purified water at room temperature demonstrated that after incubation for more than one month this drug 22 was completely stable due to the high energy needed to overcome the barrier for the cleavage of its amide 23 bond (Karaman et al. 2016). In addition, being 4-aminophenol a photo and thermal sensitive compound, like 24 other aromatic amines, the results obtained by Khan et al (2006) showed the occurrence of its autoxidation 25 after 7 days but at low rates: 0.8%, 26.6%, 20% and 13% at 50, 100, 150 and 200 mg/L, respectively. In any 26 case, the authors of both works concluded that biodegradation was more effective.

31
When paracetamol is used in excess, it can cause liver failure and necrosis due to N-Acetyl-p-benzoquinone 32 imine (NAPQI), a highly toxic paracetamol´s metabolite formed by enzymatic oxidation in the liver 33 (Bessems and Vermeulen 2001). About 30% and 55% of administered paracetamol is excreted in urine as 34 conjugates paracetamol sulfate and paracetamol glucuronide, respectively (Thomas 1993). Moreover, a 35 glutathione conjugate (1,4-Michael adduct) of NAPQI, the corresponding cysteine conjugate and 36 mercapturic acid breakdown products have also been found in urine after ingestion of paracetamol (Prescott 1 1980). NAPQI is known to be fairly unstable, however, in aqueous solution it readily hydrolyzes into 1,4-2 benzoquinone, which is another toxic metabolite (Dahlin and Nelson 1982;Snyder 2000;Bedner and 3 Maccrehan 2006). On the other hand, 4-aminophenol (the hydrolytic product of paracetamol) has highly 4 genotoxic and mutagenic effects (Majeska and Holden 1995;Yoshida et al. 1998). The potential toxic 5 effects of paracetamol in aquatic systems, mediated by its reactive oxygen species, have been reported. For

21
In the beginning of the century little was known about microbial degradation of paracetamol (Pieper and 22 Reineke, 2000). Since then, however, some advances have been made in the knowledge of this subject.

23
When biodegradation is complete (mineralization), the substances are converted to inorganic compounds 24 such as water, carbon dioxide, ammonium and nitrate, whereas partial breakdown results in the 4 demonstrated that paracetamol in activated sludge underwent biodegradation within less than one month 5 and reported that P. aeruginosa was the responsible for the biodegradation of paracetamol to 4-aminophenol 6 and hydroquinone. It is worth to note that the identification of bacteria of the genus Pseudomonas is 7 common in these various works. In fact Pseudomonas is also known for its ability to degrade other aromatic 8 compounds of environmental concern (Neumann et al. India) and thioglycolic acid was purchased from Merck (Darmstadt, Germany). All the solutions of 36 paracetamol (99% purity), hydroquinone (99% purity), 4-aminophenol (97% purity), 4-nitrophenol (99% 1 purity) were purchased from Sigma-Aldrich (Deisenhofer, Germany). Acetonitrile (ACN) and methanol, 2 both HPLC grade, and phosphoric acid (85% purity) were supplied by VWR Prolabo Chemicals (Fontenay-3 sous-Bois, France) and formic acid (HCOOH) high purity grade was obtained from Amresco (Solon, USA).

4
Phosphate buffer solution of pH = 4.88 was prepared by dissolving 4.5 g KH2PO4 and 0.0412 g 5 K2HPO4.3H2O in 500 mL of ultra pure water, using phosphoric acid (85%) to adjust pH if necessary. 6 7 2.2 Inocula source and preparation 8 Sludges from two Portuguese WWTPs with different processes were used as inocula sources. Sludge 9 collected in the Faro East WWTP's lagoon system without aeration (anaerobic sludge) was used for a first 10 set of experiments under anaerobic and moderate aerobic conditions, while sludge from the Faro Northwest 11 WWTP's oxidation ditch with aeration (aerobic sludge) was used in a second set of experiments in which 12 highly aerobic conditions were tested (Table 1).

13
To enrich the inocula from the lagoon system, 2 g of sludge sediment were inoculated on each of two 14 separate 250ml culture flasks containing 200 mL of TGM medium. In one flask 10 mL of paraffin-oil was 15 added to prevent gas diffusion and the flask was sealed with a butyl rubber stopper and an aluminum crimp 16 seal to avoid air intake in order to create anaerobic condition. In the other flask, cotton was used to serve as 17 lid, thus allowing the maintenance of aerobic conditions. To enrich the inoculum from the oxidation ditch, 2 18 mL of water with sludge was inoculated in just one flask with 200 mL TGM medium with cotton as lid. The

34
Previous steps of sludge washing were carried out to eliminate dissolved carbon compounds that could 35 serve as carbon and energy sources for bacterial growth because in some experiments (with MSM and with 36 the modified Postgate B without lactate) paracetamol was expected to be the only carbon source. With that 1 purpose, the enriched inocula coming from the anaerobic and aerobic sludges were centrifuged at 2500 × g 2 for 10 min at room temperature, the supernatant was discarded and the pellets re-suspended using the MSM.

3
This procedure was repeated twice before inoculation of test cultures.

35
One-Way ANOVA (Single Factor) tests using Excel Data Analysis Tools were performed to evaluate if 1 differences between the inoculated test cultures and the respective non inoculated negative controls were 2 significant for 5% error (α = 0.05). 3 4 2.5 Effect of paracetamol on chemical oxygen demand (COD) degradation 5 COD is often used to measure organic matter, allowing an indirect quantification of the amount of 6 oxidizable compounds in wastewaters, treated effluents and receiving waters 7 The COD (mg O2/L) was analyzed in assays with MWW medium in the presence and absence of 8 paracetamol to evaluate the influence of this drug on the degradation of the organic matter present in 9 wastewater. COD was also determined in the MSM medium assays with paracetamol (i) inoculated with 10 aerobic sludge and (ii) without inoculum, to evaluate the variation of COD caused by adding this drug and 11 by its removal. Samples collected immediately after preparation of these assays and after 72 h incubation 12 were used for COD analysis.

35
Exclusive standard calibration curves were constructed for each experiment to determine the concentrations 1 of paracetamol in the corresponding samples. The concentration ranges of paracetamol standards, prepared 2 with the respective medium were: TGM medium: 5 to 100 mg/L and 50 to 2500 mg/L, depending on the 3 paracetamol concentration tested; MSM medium: 5 to 1000 mg/L; Modified Postgate B medium: 1 to 110 4 mg/L; MWW assays: 5 to 250 mg/L. The limits of detection (LOD) were determined by the analysis of 5 standards with known concentrations to establish the minimum level at which the analyte peak could be 6 reliably detected by visual evaluation, as described in the harmonized tripartite guideline (ICH, 1996).

18
The injection volume was 20 µL, the flow rate was set at 0.8 mL/min, the column was maintained at room 19 temperature and detection was performed at 244 nm.  The results obtained clearly indicate that the major routes for the biodegradation of this drug in WWTPs 26 involve aerobic organisms; some already reported previously and others identified in this work for the first 27 time, as discussed further below. However, some slow biodegradation seems to occur under anaerobic 28 conditions, which was not more evident due to the short time of the assays.

29
The overall results obtained in the tests performed to evaluate paracetamol biodegradation under diverse 30 experimental conditions are summarized in Table 1 In the inoculated TGM cultures under anaerobic conditions with the lower tested paracetamol 7 concentrations of 10, 50 and 80 mg/L, drug removals of 24%, 22% and 12% (respectively) were observed 8 after 24 h and removals around twice these values (56%, 45% and 33%) were achieved after 72 h (Table 1).

9
With the same conditions but in the presence of 10000 mg/L paracetamol, a removal efficiency of 22±14% 10 was attained after 24 h assay, but no major changes were observed after 72 h (26±12%). According to the 11 plotted IC50 curves ( Figure 2) the bacterial viability (%) for 10000 mg/L paracetamol is low (20% to 30%).

12
Therefore, probably this high concentration inhibited the growth of bacteria degrading this drug. Even so, in 13 all these cases and for both sampling days, the differences in paracetamol removal averages between the 14 inoculated and non-inoculated tests were relatively low (< 15%) and not significant (for 5% error), 15 suggesting that biodegradation had a limited role in the removal of this drug. using modified Postgate B medium the pH remained within the interval of 6.5 to 7.5, which is in the 33 optimum range for SRB. Indeed, the parameters analyzed to monitor these bacteria indicated high activity in 34 the inoculated tests: the redox potential (Eh) evolved to values between -350 and -400 mV after 2 weeks 35 incubation under anaerobic conditions and the sulphate removal in this period was equal or higher than the 36 72% observed in the control culture without the drug. These results demonstrate that the inoculated SRB 1 consortium enriched from the Faro East WWTP's lagoon system sludge was resistant to all concentrations 2 of paracetamol tested. In what concerns the degradation of paracetamol, the results were slightly different 3 from those observed in the tests with TGM. The maximum removal of paracetamol achieved was in the 4 same range (51±2%) and the differences on the removal averages of this drug between the inoculated 5 cultures and the respective non inoculated negative controls were also small (less than 15%). However, 6 despite small, those differences were significant (for 5% error) after 14 days incubation for all paracetamol 7 concentrations tested.

8
In the assay performed using the same SRB inoculum in modified Postgate B medium without lactate and 9 spiked with 100 mg/L of paracetamol, the pH also remained neutral (7.0±0.2) in all cultures and the redox 10 potential (Eh) in the inoculated tests also evolved to values between -350 and -400 mV, which are optimal 11 conditions for SRB. However, the concentration of sulphate only decreased 25%, indicating low SRB 12 activity. In any case, the values of paracetamol removal achieved were still low (19±2%) and only slightly 13 higher than in the non inoculated negative controls (12±2%). Nevertheless, this small difference achieved 14 after 14 days of incubation was significant (for 5% error). anaerobic conditions. Experiments with MSM were first performed using sludge from the WWTP lagoon 30 system as inoculum for the anaerobic and moderate aerobic conditions. In these tests, minor (< 25%) or no 31 degradation of paracetamol was observed either after 24 or 72 h of incubation with the three concentrations 32 of paracetamol tested (10, 50, and 80 mg/L). Moreover, in these conditions the non significant (for 5% 33 error) and small differences (<10%) in paracetamol removal obtained between the negative controls and the 34 inoculated cultures, reinforce the idea that there was no degradation, or just small degradation, due to 35 biological activity, as observed in the experiments with TGM.

36
The partial removals of paracetamol achieved when the WWTP lagoon sludge was used as inoculum in 1 TGM and MSM under anaerobic and moderate aerobic conditions and the negligible contribution of the 2 inoculum in these removals suggest that the conditions of growth and/or the nature of the inoculum 3 probably hampered the biodegradation of the total amount of the drug in the tested cultures. Therefore, 4 trying to improve paracetamol biodegradation, further experiments were carried out in highly oxygenated 5 (aerated) aerobic conditions and using as inoculum sludge from a WWTP's oxidation ditch. One 6 concentration of paracetamol (50 mg/L) was tested in aerated MSM inoculated with this sludge. After 24 h 7 of incubation only 3±3% of the drug was removed, which was not significantly different (for 5% error) 8 from the 1±1% removal observed in the negative control. However, after 72 h of incubation a removal of 9 97±2% was achieved, while practically no removal (3±8%) was still observed in the non-inoculated 10 negative controls, indicating that the major cause of paracetamol removal was due to inoculation with 11 sludge. An additional test with autoclaved sludge in MSM was performed to evaluate the possible 12 adsorption of paracetamol to sludge. The results revealed just a small drug removal (15±2%), reinforcing 13 the idea that the major cause of paracetamol removal using MSM medium in highly oxygenated conditions 14 was the biological activity of aerobic microorganisms.

17
MWW was used in order to mimic the real conditions in WWTPs. It was tested just under aerobic 18 conditions, in which the main electron accepter is oxygen. Cultures with 50 mg/L paracetamol were tested 19 under moderate aerobic conditions and highly oxygenated aerobic conditions (as described above) using 20 sludge from the WWTP oxidation ditch as inoculum. After 24 h incubation just 11±10% paracetamol was 21 removed from MWW without aeration and 70±10% was removed with aeration, whereas after 72 h the 22 removal was nearly complete in both cases: 88±7% and 99.9±0.2%, respectively (Table 1). In this 23 experiment, negative controls without sludge were not carried out because the MWW is itself a source of 24 microbial inoculum, but a test with both autoclaved MWW and sludge was performed. The low paracetamol 25 removal observed in that test (6±2% and 11±5% for 24 h and 72 h incubation, respectively) is significantly 26 different from that achieved in the tests with active sludge, which indicates that biological degradation was 27 the main mechanism involved in paracetamol removal using MWW medium under highly oxygenated 28 conditions, as happened in the tests with highly oxygenated MSM described above.

31
The results suggest that paracetamol removal in the assays under anaerobic and moderate aerobic conditions 32 using sludge from the Faro East WWTP lagoon system was mainly due to other causes than microbial 33 activity: removal was always below 60% and was, in general, similar to, or weakly higher than, the obtained 34 in the respective negative controls (without microbial inoculum). Moreover, the results of the non 35 inoculated tests show higher removals for TGM (removals up to 43±12%) and modified Postgate B medium 36 (removals up to 39±5%) than for the simpler MSM medium (removals below 17±6%), suggesting that some 1 components in the most complex media were able to chemically react with paracetamol, contributing for its 2 removal. One non biological mechanism that can account for the observed losses of paracetamol in all 3 tested media is its oxidation by SO4 2ions. It is known that compared with hydroxyl radical ( • OH), SO4 2is 4 more selective for oxidation by electron-transfer reaction and more powerful for the decomposition of 5 contaminants at neutral pH (Mezyk et al. 2011;Zhang et al. 2015). Indeed, when peroxymonosulfate (PMS) 6 is activated by catalysts it decomposes generating SO4 2-, which degrades pollutants. Thus, several catalysts 7 have been explored for PMS oxidation and some attracted great interest due to its remarkable separation and

28
Though the biomass growth was not analyzed during the biodegradation tests, it was evaluated by optical 29 density measurements at the beginning and at the end of the inocula enrichment in TGM (a rich medium for 30 anaerobic and aerobic microorganisms) and during that period roughly similar growths were observed in the 31 different redox conditions (as described in section 2.2). Taking that into account, together with the fact that 32 roughly similar biomasses were inoculated in all tests, it can be assumed that at least for the tests with TGM

15
Samples from the experiments with MSM and MWW spiked with paracetamol using sludge from the 16 WWTP oxidation ditch as inoculum revealed high paracetamol removal when the HPLC analysis was 17 conducted using the fast method in isocratic conditions. Therefore, these samples were re-analyzed using an 18 HPLC gradient method that allows a good separation of the metabolites listed above, usually associated to 19 paracetamol degradation (both HPLC methods are described in materials and methods). Two peaks with 20 retention times (RT) corresponding to 4-aminophenol and hydroquinone standards, and another with a RT 21 not matching any of the tested standards (herein referred to as unknown) were detected in samples from the 22 tests in MWW and MSM inoculated with sludge ( Figure 4). The two peaks corresponding to the 23 paracetamol secondary metabolites 4-aminophenol and hydroquinone, as well as the unknown peak, 24 emerged only in samples from cultures in which the concentration of paracetamol decreased. Moreover, 25 they were neither detected at the beginning of the experiment nor in samples from the respective negative 26 controls (without inoculum). This clearly indicates that biological activity is related to the appearance of 27 these metabolites and that they are products from the degradation of paracetamol.

28
The peak corresponding to 4-aminophenol (RT = 4.1 min) and the unknown peak (RT = 8. COD is an important water quality parameter providing an index to determine the effect an effluent will 8 have on the receiving water body. It is therefore a major reference in the control of wastewater discharges.

9
For example in Portugal the general COD emission limit value for wastewater discharges is 150 mg O2/L 10 (Decree-Law nº 236/98 of 1 August) and the limit for discharges from urban WWTP is 125 mg O2/L 11 (Decree-Law nº 152/97 of 19 June).

12
In this study the effect of paracetamol on the removal of COD was also addressed. COD was measured in

35
Aiming to identify bacteria able to degrade paracetamol, thus with potential for biotechnological 1 applications to improve the removal of this drug from wastewaters, microbial communities were studied on 2 samples from the experiment with aerated cultures of inoculated MSM spiked with 50 mg/L paracetamol, in 3 which high removal rates of this drug were observed. The three replicate cultures of this test revealed 4 identical behaviors for the concentrations of the analyzed compounds along the experiment. Thus, it was 5 considered that the microbial dynamics studied through massive sequencing of 16S rRNA genes present in 6 just one of the replicates along the incubation time would provide enough robust evidences to identify taxa 7 putatively involved in the degradation of paracetamol and/or its metabolites. The study was focused on 8 bacteria using primers for the 16S rRNA gene region V1-3. Samples for microbial community analysis were 9 collected from the WWTP oxidation ditch sludge used as inoculum and from cultures with 1, 2, 3 and 6 10 days of incubation after inoculation.

31
This is not the first time that microbial populations exposed to a pharmaceutical led to shifts towards an 32 evident selection of Flavobacterium species. For example, in cultures acclimated with the antibiotic 33 enrofloxacin a selection of the genera Flavobacterium was observed with its relative abundance increasing 34 20. 8% (Alexandrino et al. 2017). In fact, by the end of the 70's it had already been described the isolation of 35 a Flavobacterium sp. capable of degrading the herbicide asulam and its structure analogous sulphanilamide 36 in a synthetic medium with no other carbon sources added (Walker, 1978). Then, the isolation of plasmids 1 carrying genes for degradation of aromatic compounds (Chaudhry and Huang, 1988)  Flavobacterium sp. able to use 4-nitrophenol as a carbon and energy source has already been isolated 10 (Raymond and Alexander, 1971) and this compound could also be used as the sole carbon and energy 11 source by a strain of P. aeruginosa (Zheng et al. 2009), a specie with strains able to biodegrade paracetamol 12 and 4-aminophenol as described above. Therefore, based on the results here presented it seems reasonable 13 to propose that probably the Flavobacterium strains in the culture corresponding to OUT_20 were capable 14 of biodegrading 4-aminophenol. The alignment of OTU_20 in the NCBI database revealed a 15 Flavobacterium luvivivi sequence with 100% coverage and 99% similarity and sequences of several species 16 of this genus with similarities of less than 95%.

17
An association between Methylophilus and successful biodegradation of pharmaceuticals using next-18 generation sequencing of 16S rRNA genes over time, besides the work here presented, was also described

32
In the genus Dokdonella 5 OTUs were identified, but also in this case only one (OTU_10; GenBank 33 accession MG554754) stood out in the evolution of relative abundances. The percentage of amplicons in 34 this OTU increased mainly in the first two days of incubation, then remained almost the same from the 35 second to the third day and after six days it increased again. Though the genus Dokdonella has not yet been 36 associated with pharmaceutical degradation it has been detected in alkane degrading cultures (Alonso-1 Gutiérrez et al. 2009) and a Dokdonella potentially degrading polycyclic aromatic hydrocarbons was 2 detected by Bacosa and Inoue (2015). Moreover, Dokdonella has been previously isolated from denitrifying 3 environments in the presence of O2 (Sun et al. 2009) and was one of the predominant microorganisms in a 4 study with activated sludge at 2% O2 concentration (without inhibitory effects on N2O biodegradation), in 5 which it was suggested that heterotrophic denitrification was the most likely mechanism of N2O removal 6 (Figueroa-González et al. 2016). It may therefore be hypothesized that the Dokdonella corresponding to 7 OTU_10 may have used the nitrate generated after the release of the amine group from 4-aminophenol (by 8 substitution by a hydroxyl group) as an electron receptor to obtain energy by oxidation of hydroquinone (the 9 aromatic organic compound generated by the amine group release of 4-aminophenol) and/or by oxidation of

Fig. 2
Graphical representation of best fitting sigmoidal curves for bacterial viability (%) as a function of logarithm of paracetamol concentration (g/L), for aerobic and anaerobic assays, with values expressed as mean ± distance to individual data points (n=2). The percentage of bacterial viability is the optical density at 600 nm (OD600) measured in a culture with a certain drug concentration divided by the OD600 in the control culture without drug, multiplied by 100. In some points the bars are smaller than the symbol, therefore not visible.