Enhanced Diclofenac Biodegradation by Bacterial Strains and a Microbial Consortium from Activated Sludge: Toxicity Assessment and Insights into Microbial Community Dynamics

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

Please find listed below my recommendations for "" manuscript:

1. L45: Please write correctly the IUPAC name for DCF
2. L45-48: This should be better considered. DCF exhibits preferential COX-2 selectivity and also inhibits COX-1, but the present form of the manuscript statement implies non-selective competitive inhibition without acknowledging isoform specificity or the time-dependent nature of inhibition
3. L59-63: This should be carefully reconsidered. Based on my knowledge, DCF does not possess a rigid bicyclic aromatic framework. It features a diphenylamine core with two separate phenyl rings connected through a secondary amine linkage - this is a flexible biaryl system, and not a bicyclic structure. A bicyclic system would require ring fusion or bridging atoms, but this is not present in  DCF. Further, the description as rigid bicyclic aromatic framework contradicts the actual conformational flexibility of the diphenylamine moiety, which exhibits rotational freedom around the C-N bonds. Also, considering the statement from L63, which degradation pathways (e.g., hydroxylation, decarboxylation, dechlorination??) are hindered and by what specific electronic or steric factors? The electron-withdrawing chlorine substituents actually activate the aromatic ring toward electrophilic attack at certain positions. I recommend for authors careful consideration for clear technical notions 
4. L73-75: How this? This need to be verified and presented in correct and clear context, such statement can not be  should not be generalized globally without qualification and specificity !!
5. L80-85: Please reconsider this statement as in its current form it seems that the authors mix general pharmaceutical toxicity mechanisms (membrane damage, lipid peroxidation, oxidative stress) with DCF-specific effects on bacteria. Please note that these are non-specific stress responses that could apply to numerous xenobiotics. The citation of catalase, superoxide dismutase, and phosphatases as "biomarkers" lacks specificity as these are ubiquitous antioxidant enzymes, not DCF-specific indicators !
6. L86-92: Here the manuscript should specify if these organisms achieve mineralization or merely biotransformation and include the enzymatic systems involved (e.g., cytochrome P450s, laccases, peroxidases). Please refer to the degradation rates or other efficiency metrics
7. L104-116: Please clearly formulate the hypothesis and proposed objectives. Please provide justification for choosing activated sludge over other environmental matrices
8. In my opinion the current form of the introduction is improper. It should be more accurate scientifically. Also, I consider that it should clear evidence the identified knowledge gaps, highlighting those that will be addressed by this research. I recommend to consider to discuss also the metabolites toxicity relative to parent compound; cometabolic vs. metabolic degradation pathways; bioaugmentation vs. biostimulation strategies; and limitations of pure culture vs. consortium approaches. These are more proper considering the manuscript than those presently addressed which are very generic, vague and imprecise statements. Please carefully reconsider the entire section
9. L142: What means "10 mL of activated sludge" in terms of OD₆₀₀, or CFU/mL, or TSS...
10. L143: Provide justification for this concentration (24 mg L⁻¹)
11. L145-152: The HPLC method should be a separate subsection. This should consider to include all relevant methods parameters and also relevant method performance metrics - recovery, LOQ, accuracy, U, etc...
12. L194-195: Why this concentrations? And control?
13. L198: How was established that 24 h is enough?
14. L201-202: The formula should be clearly stated
15. L206: Why this concentration?? 10 mg/L is 10,000-1,000,000× higher than environmental levels (ng/L to μg/L - this was declared by the manuscript in he introduction). For me the ecotoxicological relevance is questionable
16. L307-321: This should be considered in a stand alone subsection and it clearly do not belongs to statistical analysis subsection. 
17. The entire method section should be reconsidered following a logical flow pattern. The current form is chaotic and hard to understand and follow
18. I recommend for authors to reconsider carefully the experimental design. Reliable testing concentration should be considered connected with reality !!!
19. L347: What is the environmental relevance for these concentrations. For me 200 mg/L is toxicologically unrealistic and may select for resistance mechanisms rather than degradation
20. L351-355: Why no degradation progress between day 14 and day 21? This could indicate either substrate depletion/inhibition; microbial death/dormancy; or experimental failure. Mechanistic explanation for stagnation should be provided
21. L356: This amount of glucose could easily create co-metabolic conditions, DCF is not the sole carbon source as stated in L336. Glucose likely drives growth and the DCF degradation may be fortuitous/co-metabolic
22. Fig 1. Statistical interpretation should be considered 
23. Please include the confirmation that isolated colonies actually degrade DCF (Purity verification protocols should be presented in methods section, and here the results)
24. L364-367: Based on what is this stated? This is a pure speculation without experimental support
25. I recommend that the entire manuscript to be carefully reconsidered ensuring the required scientific accuracy  needed by a scientific paper

Comments on the Quality of English Language

improvements is needed

Author Response

REVIEWER 1

Dear Authors,

Please find listed below my recommendations for "" manuscript:

1. L45: Please write correctly the IUPAC name for DCF

The IUPAC name has been corrected (Line 45). The previous name was taken from Badawy et al. (2025), who stated verbatim: “DCF, chemically known as 2-{2-[(2,6-dichlorophenyl)amino]phenyl}acetic acid, is an NSAID.”

We have included the IUPAC name 2-[2-(2,6-dichloroanilino)phenyl]acetic acid, taken from PubChem (https://pubchem.ncbi.nlm.nih.gov/compound/Diclofenac#section=Names-and-Identifiers). However, we have found a different IUPAC name, 2-[(2,6-dichlorophenyl)amino]phenylacetic acid, listed by the Royal Society of Chemistry (https://www.chemspider.com/Chemical-Structure.2925.html).

References

Badawy, A.S.; Xu, Y.; Ren, H.; Lu, Z. Diclofenac (DCF) as an emerging pollutant: Occurrence, ecotoxicity, and biodegradation strategies. Ecotoxicol. Environ. Saf. 2025, 1;302:118618. https://doi.org/10.1016/j.ecoenv.2025.118618

1. L45-48: This should be better considered. DCF exhibits preferential COX-2 selectivity and also inhibits COX-1, but the present form of the manuscript statement implies non-selective competitive inhibition without acknowledging isoform specificity or the time-dependent nature of inhibition

Information about action mechanisms has been modified (Lines 45-49).

1. L59-63: This should be carefully reconsidered. Based on my knowledge, DCF does not possess a rigid bicyclic aromatic framework. It features a diphenylamine core with two separate phenyl rings connected through a secondary amine linkage - this is a flexible biaryl system, and not a bicyclic structure. A bicyclic system would require ring fusion or bridging atoms, but this is not present in  DCF. Further, the description as rigid bicyclic aromatic framework contradicts the actual conformational flexibility of the diphenylamine moiety, which exhibits rotational freedom around the C-N bonds. Also, considering the statement from L63, which degradation pathways (e.g., hydroxylation, decarboxylation, dechlorination??) are hindered and by what specific electronic or steric factors? The electron-withdrawing chlorine substituents actually activate the aromatic ring toward electrophilic attack at certain positions. I recommend for authors careful consideration for clear technical notions 

We thank the reviewer for this correction. The reviewer is absolutely correct - diclofenac does not possess a bicyclic aromatic framework. We have revised the paragraph and added new information (Lines 57-64). 

1. L73-75: How this? This need to be verified and presented in correct and clear context, such statement can not be  should not be generalized globally without qualification and specificity !!

After reviewing the information, we found that it cannot be considered specific to DCF. For this reason, we have revised the content in the introduction to indicate that both biosolids and water from wastewater treatment plants can contain DCF, representing a potential source of contamination for agricultural soils (Lines 72-73).

1. L80-85: Please reconsider this statement as in its current form it seems that the authors mix general pharmaceutical toxicity mechanisms (membrane damage, lipid peroxidation, oxidative stress) with DCF-specific effects on bacteria. Please note that these are non-specific stress responses that could apply to numerous xenobiotics. The citation of catalase, superoxide dismutase, and phosphatases as "biomarkers" lacks specificity as these are ubiquitous antioxidant enzymes, not DCF-specific indicators !

After re-reviewing the literature, we have confirmed that DCF can exert direct effects on microorganisms, as indicated in the review by Badawy et al. (2025). Specifically, the authors state: “DCF poses a significant risk to microorganisms, with potential implications for ecosystem balance and function. At high concentrations (μg/L), it can inhibit microbial growth by disrupting cell membranes, altering enzyme activities, and inducing oxidative stress through the generation of reactive oxygen species (ROS), which can damage cellular components such as lipids, proteins, and DNA (Fig. 4) (Hayes et al., 2024; Iovino et al., 2024; Lin et al., 2024).”

Indeed, the reviewer is correct. Although DCF induces the effects discussed in the text, these are not specific to this compound. Therefore, we have reconsidered the use of the term biomarkers and have modified the paragraph where it appears (Lines 82-86).

References

Badawy, A.S.; Xu, Y.; Ren, H.; Lu, Z. Diclofenac (DCF) as an emerging pollutant: Occurrence, ecotoxicity, and biodegradation strategies. Ecotoxicol. Environ. Saf. 2025, 1;302:118618. https://doi.org/10.1016/j.ecoenv.2025.118618

1. L86-92: Here the manuscript should specify if these organisms achieve mineralization or merely biotransformation and include the enzymatic systems involved (e.g., cytochrome P450s, laccases, peroxidases). Please refer to the degradation rates or other efficiency metrics

As suggested by the reviewer, we have expanded the Introduction to specify whether the microorganisms achieve mineralization or biotransformation, the enzymes involved, and the extent of biodegradation (Lines 88–122). These aspects were previously addressed in detail in the Results and Discussion section, but have now been included here to improve contextualization, as requested by the reviewer.

1. L104-116: Please clearly formulate the hypothesis and proposed objectives. Please provide justification for choosing activated sludge over other environmental matrices

Activated sludge was selected, for example, instead of raw wastewater or sludge for two main reasons. First, it represents a highly enriched and specialized microbial community shaped by continuous exposure to a broad spectrum of organic contaminants, including pharmaceuticals. Second, its origin in the secondary (biological) treatment stage of wastewater ensures that microorganisms are deliberately concentrated and maintained under aerobic conditions, favoring the biodegradation of pollutants.

In addition, activated sludge was chosen as the source material because it harbors diverse and metabolically versatile microbial communities that are routinely exposed to pharmaceuticals such as DCF. Previous studies have shown that activated sludge contains bacterial genera capable of degrading aromatic compounds and pharmaceuticals, and that chronic exposure to pharmaceutical residues in wastewater treatment plants promotes the adaptation and selection of microorganisms with the capacity to transform these contaminants (Kraigher et al., 2008; Nguyen et al., 2019). The activated sludge used in this study receives effluents from sources including the Virgen Macarena University Hospital (Seville) and several municipalities in the province (Lines 179–180), further supporting its suitability for this work

For clarity, this information has been revised in the manuscript (Lines 146-161) as follows:

“Activated sludge, collected from aeration tanks, was selected as a rich and diverse microbial reservoir since WWTPs are often exposed to pharmaceutical compounds [39], imposing selective pressure on the bacterial community, potentially selecting microorganisms with the ability to survive in the presence of these compounds. Based on this, the objectives of the study were: i) to establish enrichment cultures from activated sludge to obtain microbial consortia and bacterial isolates capable of degrading DCF; ii) to evaluate DCF degradation efficiency in aqueous solutions and identify the metabolites produced during the biodegradation process; iii) to assess the ecotoxicological impact of DCF before and after biodegradation, thereby evaluating the effectiveness of the decontamination strategy in terms of toxicity mitigation and iv) to explore microbial community dynamics during DCF degradation using 16S rRNA and ITS amplicon sequencing, to identify the key bacterial and fungal taxa potentially involved in DCF biotransformation. This study offers interdisciplinary insights by integrating microbiology, analytical chemistry, toxicology, and metabarcoding to advance the understanding of DCF biodegradation, a contaminant that remains insufficiently characterized in terms of its microbial degraders, transformation pathways, and associated toxicity.”

References

Nguyen LN, Nghiem LD, Pramanik BK, Oh S. Cometabolic biotransformation and impacts of the anti-inflammatory drug diclofenac on activated sludge microbial communities. Sci Total Environ. 2019 Mar 20;657:739-745. doi: 10.1016/j.scitotenv.2018.12.094.

Kraigher B, Kosjek T, Heath E, Kompare B, Mandic-Mulec I. Influence of pharmaceutical residues on the structure of activated sludge bacterial communities in wastewater treatment bioreactors. Water Res. 2008 Nov;42(17):4578-88. doi: 10.1016/j.watres.2008.08.006.

1. In my opinion the current form of the introduction is improper. It should be more accurate scientifically. Also, I consider that it should clear evidence the identified knowledge gaps, highlighting those that will be addressed by this research. I recommend to consider to discuss also the metabolites toxicity relative to parent compound; cometabolic vs. metabolic degradation pathways; bioaugmentation vs. biostimulation strategies; and limitations of pure culture vs. consortium approaches. These are more proper considering the manuscript than those presently addressed which are very generic, vague and imprecise statements. Please carefully reconsider the entire section

We thank the reviewer for this constructive comment. The Introduction has been carefully revised to improve its scientific accuracy and to better highlight the relevant knowledge gaps addressed in this study.

These aspects suggested by the reviewer have been briefly introduced to provide appropriate context and to clearly frame the objectives of the present work, while avoiding an excessive level of detail that would extend beyond the scope of the Introduction. Many of these topics are discussed in depth in the Results and Discussion sections, in direct relation to the experimental findings. Furthermore, the length of the Introduction has already increased considerably following the incorporation of suggestion 6.

The content included can be found in the following Lines:

Bioaugmentation and biostimulation: Lines 74-80 and 91-122.

Metabolic degradation pathways: Lines 88-91.

Cometabolism: Lines 112-122.

Pure cultures versus microbial consortia: Lines 123-131.

Toxicity: Lines 142-145.

1. L142: What means "10 mL of activated sludge" in terms of OD₆₀₀, or CFU/mL, or TSS...

As indicated in Section 2.2.1, 10 mL of activated sludge was used to perform the enrichment cultures aimed at obtaining DCF-specific degrading microorganisms. Measuring the OD₆₀₀ of activated sludge is not meaningful, as it is a complex matrix containing a high amount of organic matter. Regarding CFU/mL, this was not determined in the present study, as we considered it irrelevant given that the sample was freshly taken from WWTPs, which are known to harbor a high abundance of microorganisms. Even more, the initial quantity of microorganisms present is not a critical factor in enrichment cultures in order to isolate a potential contaminant degrader. This same procedure has been successfully applied in our previous studies using environmental matrices such as sewage sludge (Aguilar-Romero et al., 2025) and soils (Lara-Moreno et al., 2022), without prior knowledge of the CFU/mL in the initial samples.

References

Aguilar-Romero, I.; Madrid, F.; Villaverde, J.; Alonso, E.; Santos, J.L.; Morillo, E. Removal of Ibuprofen in Water by Bioaugmentation with Labrys neptuniae CSW11 Isolated from Sewage Sludge—Assessment of Biodegradation Pathway Based on Metabolite Formation and Genomic Analysis. J. Xenobiot. 2025, 15, 5. https://doi.org/10.3390/jox15010005

Lara-Moreno A, Morillo E, Merchán F, Madrid F, Villaverde J. Bioremediation of a trifluralin contaminated soil using bioaugmentation with novel isolated bacterial strains and cyclodextrin. Sci Total Environ. 2022 Sep 20;840:156695. doi: 10.1016/j.scitotenv.2022.156695.

1. L143: Provide justification for this concentration (24 mg L⁻¹)

We decided to base our method on the protocol described by Bessa et al. (2017), who used activated sludge to isolate DCF-degrading microorganisms in the presence of 24 mg L⁻¹ of DCF. Lines 184-185 indicates that we followed the protocol established by these authors. Additionally, we included the information (Lines 188-191): "DCF concentration used for enrichment cultures exceeds the concentrations typically observed in WWTPs, to create a selective pressure on the bacterial community, facilitating the enrichment and isolation of strains capable of degrading this compound", reported by Bessa et al. (2017).

Moreover, based on our experience, exposing an environmental matrix to different pharmaceutical concentrations in enrichment cultures promotes the selection of distinct degrading microorganisms, thereby increasing the range of potential success in the search for specific degraders. Indeed, in our study, a concentration of 24 mg L⁻¹ led to the isolation of the MC consortium (Lines 484-485) and the strain CSWD.2 (Lines 493-494), whereas at a concentration of 200 mg L⁻¹, the specific degrading strain CSWD.1 was obtained (line 492-493). Hence, we confirm that this concentration-based approach is appropriate, as it enabled the isolation of specific degrading microorganisms.

References

Bessa, V.S.; Moreira, I.S.; Tiritan, M.E.; Castro, P.M.L. Enrichment of bacterial strains for the biodegradation of diclofenac and carbamazepine from activated sludge. Int. Biodeterior. Biodegrad. 2017, 120, 135–142. https://doi.org/10.1016/j. ibiod.2017.02.008.

1. L145-152: The HPLC method should be a separate subsection. This should consider to include all relevant methods parameters and also relevant method performance metrics - recovery, LOQ, accuracy, U, etc..

HPLC method information has been moved to a new section, “2.2.5. Analytical Methods.”. Nevertheless, as we have used a method previously well established (Dorado et al., 2023), parameters such as Uncertainty were not calculated. Quantification limit was calculated with blank samples, an external method calibration (linearity was checked with R²), and the standard deviation of samples was calculated from triplicates. On the other hand, as all samples were liquid, recovery was not necessary.

Reference

Dorado, P.; Berecz, R.; Cáceres, M.C.; Llerena, A. Analysis of diclofenac and its metabolites by high-performance liquid chromatography: Relevance of CYP2C9 genotypes in diclofenac urinary metabolic ratios. J. Chromatogr. B. 2003, 789(2):437–42. https://doi.org/10.1016/s1570-0232(03)00137-5.

1. L194-195: Why this concentrations? And control?

The DCF concentrations (10, 100, 500, 1000, 3000, and 5000 mg L⁻¹) were selected to cover a wide range, from low levels to high levels considered potentially toxic, thereby facilitating an accurate determination of the IC₅₀ (This information has been included in the manuscript: Lines 239-240). This selection was supported by previous studies that commonly use similar concentration ranges to observe microbial responses to pharmaceuticals (Aguilar-Romero et al., 2025; Vargas-Ordoñez et al., 2023). In addition, parallel control without DCF was included to represent normal microbial growth and to rule out any issues related to the inoculum, culture medium, or other experimental conditions.

References

Aguilar-Romero, I.; Lara-Moreno, A.; Madrid, F.; Villaverde, J.; Alonso, E.; Santos, J.L.; Morillo, E. Removal of Ibuprofen from Contaminated Water by Bioaugmentation with Novel Bacterial Strains Isolated from Sewage Sludge. Microorganisms 2025, 13, 1927. https://doi.org/10.3390/microorganisms13081927

Vargas-Ordóñez, A.; Aguilar-Romero, I.; Villaverde, J.; Madrid, F.; Morillo, E. Isolation of Novel Bacterial Strains Pseudomonas extremaustralis CSW01 and Stutzerimonas stutzeri CSW02 from Sewage Sludge for Paracetamol Biodegradation. Microorganisms 2023, 11, 196. https://doi.org/10.3390/microorganisms11010196

13. L198: How was established that 24 h is enough?

The 24 h exposure time was selected based on bacterial growth kinetics. Growth curves of the bacterial strains CSWD.1 and CSWD.2 are included in the Supplementary Material (Figure S1) and show that both strains reach the stationary phase after approximately 15 h. Therefore, the selected exposure time of 24 h ensures that bacterial growth has stabilized before endpoint determination. In the case of the consortium, a 24 h exposure time was also chosen to allow direct comparison with the growth curves and to ensure consistency in the IC₅₀ determination.

In addition, this exposure period is consistent with established practice in IC₅₀ and growth inhibition assays, where incubation times of 18–24 h are widely accepted (CLSI, 2018). Under the experimental conditions applied, bacteria undergo multiple generations within 24 h, which is sufficient for the compound to exert measurable inhibitory effects on growth. Longer exposure times may introduce confounding factors, such as nutrient depletion or the accumulation of metabolic by-products, potentially interfering with the accurate determination of IC₅₀ values. Moreover, a 24 h incubation period is commonly used and recommended in standardized bacterial toxicity and susceptibility assays, thereby facilitating comparison with previous studies (e.g., Matejczyk et al., 2020; Aguilar-Romero et al., 2025; Vargas-Ordoñez et al., 2023).

The justification has been included in (Lines 519-522).

References

Clinical and Laboratory Standards Institute (CLSI).
Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically. CLSI standard M07. Wayne, PA: CLSI; 2018.

Matejczyk M, Ofman P, Dąbrowska K, Świsłocka R, Lewandowski W. Synergistic interaction of diclofenac and its metabolites with selected antibiotics and amygdalin in wastewaters. Environ Res. 2020 Jul;186:109511. doi: 10.1016/j.envres.2020.109511.

Aguilar-Romero, I.; Lara-Moreno, A.; Madrid, F.; Villaverde, J.; Alonso, E.; Santos, J.L.; Morillo, E. Removal of Ibuprofen from Contaminated Water by Bioaugmentation with Novel Bacterial Strains Isolated from Sewage Sludge. Microorganisms 2025, 13, 1927. https://doi.org/10.3390/microorganisms13081927

Vargas-Ordóñez, A.; Aguilar-Romero, I.; Villaverde, J.; Madrid, F.; Morillo, E. Isolation of Novel Bacterial Strains Pseudomonas extremaustralis CSW01 and Stutzerimonas stutzeri CSW02 from Sewage Sludge for Paracetamol Biodegradation. Microorganisms 2023, 11, 196. https://doi.org/10.3390/microorganisms11010196

14. L201-202: The formula should be clearly stated

The information about the calculation of IC₅₀ has been clarified (Lines 245-254):

The IC₅₀ values were calculated from the dose–response curves generated by plotting microbial viability (%) against the logarithm of diclofenac concentration (mg L⁻¹). Each dataset was fitted with a second-order polynomial regression of the form:

where y represents microbial viability (%), log[DCF] is the logarithm of the diclofenac concentration (mg L⁻¹), and a, b, and c are the experimentally determined regression coefficients. To determine IC₅₀, a value of y = 50 was substituted into the equation, the corresponding log[DCF] value was calculated, and the antilogarithm was applied to obtain the diclofenac concentration associated with 50% growth inhibition.

1. L206: Why this concentration?? 10 mg/L is 10,000-1,000,000× higher than environmental levels (ng/L to μg/L - this was declared by the manuscript in he introduction). For me the ecotoxicological relevance is questionable

10 mg/L is indeed higher than typical environmental concentrations. However, we have demonstrated that the bacterium Pseudomonas sp. (P. syringae in the previous version of the manuscript) and microbial consortium used in this study were able to completely degrade even high DCF concentrations in solution. Moreover, several previous studies have employed similar or even higher diclofenac concentrations. For example, Poddar et al. (2024) used 500 mg/L in their biodegradation studies; Ivshina et al. (2019) used 50 mg/L; Stylianou et al. (2018) used 70 mg/L—all of which are higher than the concentrations used in our work. Additionally, Bessa et al. (2017) employed the same concentration as our study, 10 mg/L. Using higher concentrations in laboratory studies is common practice to ensure measurable biodegradation and to assess the capabilities of microorganisms under controlled conditions.

However, we acknowledge that this should be clearly indicated in the manuscript. Therefore, the following sentence has been included:

“The concentration used for the biodegradation studies in the present work is higher than those found in WWTPs and other environmental matrices, to ensure measurable biodegradation and assess the capabilities of microorganisms under controlled conditions. Other authors followed similar approaches (Bessa et al., 2017; Stylianou et al., 2018; Ivshina et al., 2019; Poddar et al., 2024).” Lines 615-619.

References

Stylianou, K.; Hapeshi, E.; Vasquez, M.I.; Fatta-Kassinos, D.; Vyrides, I. Diclofenac Biodegradation by Newly Isolated Klebsiella Sp. KSC: Microbial Intermediates and Ecotoxicological Assessment. J. Environ. Chem. Eng. 2018, 6, 3242–3248. https://doi.org/10.1016/j.jece.2018.04.052

Ivshina, I.B.; Tyumina, E.A.; Kuzmina, M.V.; Vikhareva, E.V. Features of diclofenac biodegradation by Rhodococcus ruber IEGM 346. Sci. Rep. 2019, 9, 1–13. https://doi.org/10.1038/s41598-019-45732-9.

Poddar, K.; Sarkar, D.; Bhoi, R.; Sarkar, A. Biotransformation of diclofenac by isolated super-degrader Pseudomonas sp. DCα4: Postulated pathways, and attenuated ecotoxicological effects. Environ. Pollut. 2024, 344, 123388. https://doi.org/10.1016/j.envpol.2024.123388.

Bessa, V.S.; Moreira, I.S.; Tiritan, M.E.; Castro, P.M.L. Enrichment of bacterial strains for the biodegradation of diclofenac and carbamazepine from activated sludge. Int. Biodeterior. Biodegrad. 2017, 120, 135–142. https://doi.org/10.1016/j. ibiod.2017.02.008.

1. L307-321: This should be considered in a stand alone subsection and it clearly do not belongs to statistical analysis subsection. 

Information on kinetic models has been added to the section DCF Biodegradation Test in Solution (Lines 278-295), and the statistical analyses of DNA metabarcoding have been relocated to Section 2.2.7, Characterization of Microbial Consortium by DNA Metabarcoding (Lines 384-397).

1. The entire method section should be reconsidered following a logical flow pattern. The current form is chaotic and hard to understand and follow

After addressing the previous comments, clarifying the confusing points, and moving the contents of different methodological subsections, we consider that the structure of the whole method section is now the appropriate to allow the right understanding and reading flow of the readers.

The methodological workflow followed in this study is presented as follows:

1. Enrichment and isolation of DCF-degrading consortium and DCF-degrading strains.
2. Molecular identification of bacterial isolates.
3. Determination of the inhibitory concentration of DCF for bacterial growth.
4. DCF biodegradation test in solution.
5. Analytical methods – placed as a separate subsection following the reviewer’s suggestion. In the original version, these methods were embedded within subsection 4, as this was the first point at which DCF quantification was required.
6. Ecotoxicity bioassays – performed after completing the biodegradation tests to evaluate whether the metabolites formed during DCF transformation exhibited toxicity.
7. Characterization of the microbial consortium by DNA metabarcoding
8. Statistical analysis – this subsection has been removed. For greater clarity, the statistical procedures are now described directly within each methodological subsection where they were applied.

1. I recommend for authors to reconsider carefully the experimental design. Reliable testing concentration should be considered connected with reality !!!

Thank you for this recommendation. We agree that working with environmentally relevant concentrations is important, and we will consider this in future studies. However, our primary objective in this work was to demonstrate that some of the isolated bacteria were indeed capable of degrading DCF, and this required using concentrations that allowed us to clearly detect and quantify biodegradation. Our goal was demonstrated with the concentrations used.

In addition, the Inhibitory Concentration of DCF for Bacterial Growth assay was specifically performed to ensure that the isolates selected for the study were not inhibited by the higher DCF concentrations used in the biodegradation experiments. This is precisely why we were able to employ those concentrations without compromising bacterial growth or the validity of the assays.

For future work, once the degradative potential of the isolates has been established, we plan to evaluate also their performance under lower, environmentally relevant DCF concentrations to better reflect real-world scenarios.

1. L347: What is the environmental relevance for these concentrations. For me 200 mg/L is toxicologically unrealistic and may select for resistance mechanisms rather than degradation

We have not used 200 mg/L for remediation tests. Still, only for enrichment cultures to isolate specific DCF-degrading microorganisms, so no toxicological or environmental relevance has to be taken into account here. The use of high concentrations in this step facilitates the selection and proliferation of certain microbial taxa that can utilize the contaminant as their sole carbon source. In contrast, other taxa gradually disappear during successive enrichments because they lack the metabolic tools to degrade it.

20. L351-355: Why no degradation progress between day 14 and day 21? This could indicate either substrate depletion/inhibition; microbial death/dormancy; or experimental failure. Mechanistic explanation for stagnation should be provided

Several factors may explain the observed plateau in DCF biodegradation in Figure 1 between day 14 and day 21.

One possible explanation is substrate inhibition and toxicity effects. As discussed throughout the manuscript, DCF and some of its transformation products can induce oxidative stress and cellular damage in microorganisms, thereby reducing their metabolic activity and degradative capacity. DCF has been shown to alter microbial membrane structure, increase membrane rigidity, and promote lipid peroxidation, ultimately decreasing microbial viability and inhibiting biodegradation processes (Gröning et al., 2007; Żur et al., 2020, 2021). In addition, the accumulation of transformation products that are more toxic than the parent compound may contribute to the stagnation of degradation. Reactive metabolites such as p-benzoquinone imines and 5-OH-DCF are highly electrophilic and can covalently bind to cellular proteins, impairing microbial enzymatic activity (Tang, 2003; Gröning et al., 2007). Similarly, 4-OH-DCF, formed during DCF hydroxylation, has been reported to be more harmful than DCF itself (Matejczyk et al., 2020). Furthermore, DCF-benzoic acid has been identified as a persistent end-product resistant to further biodegradation, which may accumulate and limit overall mineralization (Wu et al., 2019). The formation of nitro-diclofenac has also been associated with increased toxicity in ecotoxicological assays, potentially inhibiting microbial activity (Osorio et al., 2016).

Another contributing factor may be nutrient depletion. Enrichment cultures initially contain higher levels of organic matter due to the presence of activated sludge; however, over time this organic matter can be depleted, leading to reduced microbial activity or entry into a dormant state. Under such conditions, DCF degradation may become limited by the availability of additional carbon or energy sources. This interpretation is supported by our observation that the addition of glucose or yeast extract as co-substrates reactivated the biodegradation process. Indeed, several studies included in the manuscript report that DCF biodegradation is significantly enhanced under cometabolic conditions (Aissaoui et al., 2016; Żur et al., 2020, 2021; Papai et al., 2024).

Overall, the stagnation observed between day 14 and day 21 is most likely the result of a combination of toxic effects of DCF and its metabolites, accumulation of recalcitrant transformation products, and nutrient limitation, rather than experimental failure.

To justify this halt in biodegradation, additional information has been included in the manuscript (Lines 419-426).

References

Aissaoui, S.; Ouled-Haddar, H.; Sifour, M.; Harrouche, K.; Sghaier, H. Metabolic and co-metabolic transformation of diclofenac by Enterobacter hormachei D15 isolated from activated sludge. Curr. Microbiol. 2016, 74, 381–388. https://doi.org/10.1007/s00284-016-1190-x.

Gröning, J.; Held, C.; Garten, C.; Claussnitzer, U.; Kaschabek, SR.; Schlömann, M. Transformation of diclofenac by the indigenous microflora of river sediments and identification of a major intermediate. Chemosphere. 2007, 69(4):509-16. https://doi.org/10.1016/j.chemosphere.2007.03.037

Matejczyk, M.; Ofman, P.; Dąbrowska, K.; Świsłocka, R.; Lewandowsk, W. Evaluation of the biological impact of the mixtures of diclofenac with its biodegradation metabolites 4’-hydroxydiclofenac and 5-hydroxydiclofenac on Escherichia coli. DCF synergistic effect with caffeic acid. Arch. Environ. Protect. 2020, 46,4, 1-22. https://doi.org/10.24425/aep.2020.135760

Osorio, V.; Sanchís, J.; Abad, JL.; Ginebreda, A.; Farré, M.; Pérez, S.; Barceló, D. Investigating the formation and toxicity of nitrogen transformation products of diclofenac and sulfamethoxazole in wastewater treatment plants. J. Hazard. Mat. 2016, 309, 157-164. https://doi.org/10.1016/j.jhazmat.2016.02.013

Pápai, M.; Benedek, T.; Sörös, C.; Háhn, J.; Csenki, Z.; Bock, I.; Táncsics, A.; Kriszt, B. Biotransformation of diclofenac by Stenotrophomonas humi strain DIC_5 and toxicological examination of the resulting metabolites. Appl. Microbiol. Biotechnol. 2024, 11;108(1):485. https://doi.org/10.1007/s00253-024-13320-1.

Tang W. The metabolism of diclofenac--enzymology and toxicology perspectives. Curr Drug Metab. 2003 Aug;4(4):319-29. https://doi.org/10.2174/1389200033489398.

Wu G, Geng J, Li S, Li J, Fu Y, Xu K, Ren H, Zhang X. Abiotic and biotic processes of diclofenac in enriched nitrifying sludge: Kinetics, transformation products and reactions. Sci Total Environ. 2019 Sep 15;683:80-88. https://doi.org/10.1016/j.scitotenv.2019.05.216.

Żur, J.; Marchlewicz, A.; Piński, A.; Guzik, U.; Wojcieszyńska, D. Degradation of diclofenac by new bacterial strains and its influence on the physiological status of cells. J. Hazard. Mater. 2021, 5, 403:124000. https://doi.org/10.1016/j.jhazmat.2020.124000

Żur, J.; Piński, A.; Wojcieszyńska, D.; Smułek, W.; Guzik, U. Diclofenac Degradation-Enzymes, Genetic Background and Cellular Alterations Triggered in Diclofenac-Metabolizing Strain Pseudomonas moorei KB4. Int. J. Mol. Sci. 2020, 16;21(18):6786. https://doi.org/10.3390/ijms21186786.

1. L356: This amount of glucose could easily create co-metabolic conditions, DCF is not the sole carbon source as stated in L336. Glucose likely drives growth and the DCF degradation may be fortuitous/co-metabolic

Line 356 indicates that diclofenac is the sole carbon source, as no other carbon sources are added at the beginning of the enrichment. However, glucose and yeast extract are added at day 21. To avoid confusion, we have included the word added (Line 401), since the activated sludge already contains some carbon sources.

1. Fig 1. Statistical interpretation should be considered 

Statistical interpretation has been considered. The legend of Figure 1 has been modified: “Figure 1. Evolution of the residual percentage of DCF (24 mg L^-1 initial concentration) during enrichment culture, without activated sludge (CONTROL), with activated sludge in the absence of external carbon sources (AS). After 21 days, AS was split into three: i) AS, ii) in the presence of 3 g L^-1 glucose (AS + GLU), and iii) in the presence of 3 g L^-1 yeast extract (AS + YE). Error bars indicate standard deviation from the replicates. t-test was applied for statistical differences in times 0, 7, 14 and 21. * means significant differences among treatments Control and AS (p<0.01). ANOVA was used for times 28 and 35. Different letters for each bar mean significant differences among the corresponding treatments (Tukey test, significance level 99%).”

23. Please include the confirmation that isolated colonies actually degrade DCF (Purity verification protocols should be presented in methods section, and here the results)

To obtain pure cultures of the DCF-degrading bacteria, several successive streakings were performed until pure isolated colonies were obtained, as has been clarified in lines 208-212. Each isolated strain was then tested in biodegradation assays in solution in the presence of DCF. Strains showing promising degradation results were selected for 16S rRNA gene sequencing, which confirmed the purity of the cultures coming from single bacterial species. The sequencing procedure is detailed in section 2.2.2.

1. L364-367: Based on what is this stated? This is a pure speculation without experimental support

This statement, “In bacterial systems, glucose can promote microbial growth, which in turn intensifies DCF degradation through the activation of key enzymes such as cytochrome P-450, catechol 1,2-dioxygenase, and protocatechuate dioxygenases, among others. This results in increased oxidation, hydroxylation, and aromatic ring cleavage, processes that are essential for DCF biodegradation. “ is based on information extracted from the results of the article: Żur, J.; Piński, A.; Wojcieszyńska, D.; Smułek, W.; Guzik, U. Diclofenac Degradation—Enzymes, Genetic Background and Cellular Alterations Triggered in Diclofenac-Metabolizing Strain Pseudomonas moorei KB4. Int. J. Mol. Sci. 2020, 21, 6786. https://doi.org/10.3390/ijms21186786. However, since our study does not include experimental evidence to support it, we have decided to remove this statement.

1. I recommend that the entire manuscript to be carefully reconsidered ensuring the required scientific accuracy  needed by a scientific paper

We sincerely thank the reviewer for such a complete review, including the last recommendation. Therefore, we have carefully revised the entire manuscript to ensure scientific accuracy and clarity, checking all statements, data interpretations, and references to meet the standards required for a scientific publication.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript reports enrichment of diclofenac (DCF)-degrading microorganisms from activated sludge, isolation of two bacterial strains (reported as Pseudomonas aeruginosa CSWD.1 and Pseudomonas syringae CSWD.2), and a mixed microbial consortium (MC). In MSM supplemented with glucose (3 g L⁻¹), CSWD.2 and the MC achieved complete removal of 10 mg L⁻¹ DCF within 21 and 5 days, respectively, whereas CSWD.1 reached ~45.5% removal after 28 days. The authors monitor selected transformation products (notably hydroxylated DCF and a nitrated TP termed TP339/NO₂-DCF), assess acute toxicity using Microtox (Aliivibrio fischeri), and describe microbial community shifts (16S/ITS2 amplicon sequencing) in the MC between the beginning and end of incubation. The overall message is that DCF removal does not necessarily equate to detoxification.

General comments

Strengths

• Clear applied motivation (DCF as an emerging contaminant; need for sustainable removal).

• Useful multi-pronged approach combining kinetics, targeted metabolite screening, Microtox toxicity, and microbial community profiling.

• Triplicate biodegradation assays and kinetic modelling (SFO/HS) are appropriate for the degradation component.

Major concerns requiring substantial revision

1. Amplicon-sequencing inference is not statistically supported (n = 1 per timepoint).
   The community analysis compares only two MC samples (start vs end). Without biological replication, claims of “selection/positive selection” and any functional interpretation (e.g., specific taxa driving DCF transformation or “using DCF as carbon source”) are not justified. These statements should be substantially toned down or the dataset expanded with replicates.

2. Supplementary chemical-structure table (Table S3) appears internally inconsistent and likely contains errors.
   Several entries (formulas/weights/structures) appear chemically inconsistent with diclofenac transformation chemistry. This undermines confidence in TP nomenclature used in the main text. Table S3 must be carefully verified and corrected.

3. Metabolite evidence and quantification for TP339 (NO₂-DCF) are insufficient to support strong toxicity attribution.
   TP339 is tracked via MRM conditions referenced to prior literature, without reporting confirmation by standards/HRMS, and results appear semi-quantitative (peak areas). Yet the discussion attributes increased toxicity to specific metabolites. This needs clearer qualification, improved confirmation, and/or more cautious conclusions.

4. Biosafety and feasibility are not adequately addressed.
   One isolate is reported as P. aeruginosa (opportunistic pathogen). Any implication of practical bioaugmentation should include biosafety/regulatory considerations, or the strain should be repositioned as a model organism for pathway discovery rather than a candidate for application.

5. Environmental relevance of the experimental conditions needs stronger framing.
   The use of 10 mg L⁻¹ DCF and glucose at 3 g L⁻¹ reflects an enrichment/high-load scenario rather than typical environmental conditions. The manuscript should explicitly state this limitation and avoid overgeneralizing to real wastewater or surface-water concentrations without supporting tests.

Specific comments 

Major revisions

1. Replication and interpretation of metabarcoding

   • Line 753–758: Only two MC samples are compared (“beginning and end”). Please either (i) add biological replicates for sequencing (recommended), or (ii) explicitly label the sequencing results as descriptive/hypothesis-generating and remove/soften causal language (e.g., “selection”, “positive selection”, “using DCF as carbon source”).

   • Line 800–806: “Positive selection” wording is too strong without replication. Rephrase to “increased in relative abundance during the incubation”.

   • Line 324–331: Rarefaction depths differ strongly between bacterial and fungal datasets. Please discuss potential bias from low fungal reads and provide read statistics/rarefaction curves (main or supplement).

2. Taxonomic identification of isolates

   • Line 179–188: Identification relies on 16S rRNA BLASTn. For Pseudomonas, 16S often lacks species-level resolution. Please provide accession numbers and either support species-level identification with additional markers (e.g., gyrB, rpoD)/genome-based methods, or revise to Pseudomonas sp. where not robust.

3. Biosafety and application claims

   • Line 15–19 (Abstract) and Conclusions (Line 911–917): Given the reported P. aeruginosa isolate, explicitly discuss biosafety limitations for application and/or reframe the isolate as a laboratory model. Avoid language implying direct deployment without addressing risks.

4. Metabolite identification/quantification and toxicity attribution

   • Line 254–260: Clearly indicate which analytes were quantified with authentic standards versus screened without standards (semi-quantitative, peak area only).

   • Line 725–729 & 731–744: Temper claims linking toxicity to NO₂-DCF and hydroxylated metabolites. Add explicit uncertainty statements (e.g., “tentative identification”, “relative signal only”, “no absolute concentration”).

   • Line 737–744: There appears to be a strain-label inconsistency: TU = 10.5 is attributed differently across text and conclusions. Please correct and ensure consistency throughout.

5. Supplementary Table S3 accuracy

   • Table S3: Verify and correct all formulas, molecular weights, and structures; correct terminology (see “nitrification” note below). Ensure TP naming is consistent between main text, figures, and supplementary material.

6. Environmental relevance / framing

   • Line 498–500 and Methods Line 205–206: Add a short justification/limitations statement about using glucose (3 g L⁻¹) and 10 mg L⁻¹ DCF, clearly positioning the study as enrichment/high-load and not directly representative of typical environmental conditions.

Minor revisions 

7. Template text left in manuscript

   • Line 907–909: Delete the sentence “This section may be divided by subheadings…”, which appears to be leftover template guidance.

8. Figure caption inconsistency

   • Line 843–846 (Figure 6 caption): Caption text appears to refer to bacterial genera when the figure is discussed as fungal. Please correct caption wording to match the actual figure content.

9. Terminology

   • Table S3: Replace “nitrification” with “nitration”/“nitrosation” (nitrification is a microbial nitrogen-cycle process, not a chemical substitution on the DCF ring).

10. Microtox method clarity

• Line 262–273: Please clarify controls for matrix effects (pH, salinity, MSM components, glucose) and confirm that abiotic controls underwent the same incubation. Reporting pH at sampling (or stating adjustment) would strengthen interpretability.

11. Data availability statement

• Line 293–295: Ensure the sequencing accession information is complete and that raw reads plus sample metadata are deposited. Confirm that sample IDs match those used in supplementary ASV tables/legends.

Author Response

REVIEWER 2

The manuscript reports enrichment of diclofenac (DCF)-degrading microorganisms from activated sludge, isolation of two bacterial strains (reported as Pseudomonas aeruginosa CSWD.1 and Pseudomonas syringae CSWD.2), and a mixed microbial consortium (MC). In MSM supplemented with glucose (3 g L⁻¹), CSWD.2 and the MC achieved complete removal of 10 mg L⁻¹ DCF within 21 and 5 days, respectively, whereas CSWD.1 reached ~45.5% removal after 28 days. The authors monitor selected transformation products (notably hydroxylated DCF and a nitrated TP termed TP339/NO₂-DCF), assess acute toxicity using Microtox (Aliivibrio fischeri), and describe microbial community shifts (16S/ITS2 amplicon sequencing) in the MC between the beginning and end of incubation. The overall message is that DCF removal does not necessarily equate to detoxification.

General comments

Strengths

• Clear applied motivation (DCF as an emerging contaminant; need for sustainable removal).
• Useful multi-pronged approach combining kinetics, targeted metabolite screening, Microtox toxicity, and microbial community profiling.
• Triplicate biodegradation assays and kinetic modelling (SFO/HS) are appropriate for the degradation component.

Major concerns requiring substantial revision

1. Amplicon-sequencing inference is not statistically supported (n = 1 per timepoint).
   The community analysis compares only two MC samples (start vs end). Without biological replication, claims of “selection/positive selection” and any functional interpretation (e.g., specific taxa driving DCF transformation or “using DCF as carbon source”) are not justified. These statements should be substantially toned down or the dataset expanded with replicates.

We agree with the reviewer´s comment on the statistical limitation of the lack of replicates; however, DNA metabarcoding analyses involve considerable financial costs. Therefore, we selected the two most representative sampling time points from the DCF biodegradation experiment (“start and end” named in the manuscript and figures as “before” and “after”) to address some of the main objectives of this study, namely, identifying the bacterial and fungal genera involved in DCF biodegradation. To ensure that the number of selected samples was acceptable, we conducted an exhaustive review of similar studies in the literature. For example, Schaerer et al. (2024), Papai et al. (2023), and Demaria et al. (2025) used single samples (n = 1) for microbial community detection. Regarding sampling times, we have observed that in more complex matrices than water, such as soils or sludge, many studies examine community changes only at the beginning and at the end of the experiment. For instance, Davids et al. (2017) analyzed the bacterial community in activated sludge exposed to different ibuprofen concentrations, sampling only at the start and after 21 days, while Dou et al. (2021) investigated the bacterial community during phenanthrene biodegradation in soil, sampling at the initial time and after 25 days once the contaminant was fully degraded.

Nevertheless, we acknowledge the limitations of this approach, and in future studies, we aim to include biological triplicates and at least one intermediate sampling point during the biodegradation process. Following the reviewer´s suggestion, we have toned down the mentioned statements using a different language (i.e., avoiding the terms “selection”, “positive selection”, “using DCF as carbon source”– see more details in the response to other related comments below.

References

Davids, M., Gudra, D., Radovica-Spalvina, I., Fridmanis, D., Bartkevics, V., Muter, O. 2017. The effects of ibuprofen on activated sludge: Shift in bacterial community structure and resistance to ciprofloxacin. J. Hazard. Mater. 340, 291-299. Doi: 10.1016/j.jhazmat.2017.06.065

Demaria F, Blattner R, Puorger C, Kolvenbach B, Cretoiu MS, Hettich T, Corvini P, Lipps G, Suleiman M. Thermophilic compost bacteria as a promising approach for removal of diclofenac and related pharmaceuticals from wastewater. Water Res. 2026 Jan 1;288(Pt A):124629. doi: 10.1016/j.watres.2025.124629.

Dou, R., Sun, J., Lu, J., Deng, F., Yang, C., Lu, G., Dang, Z. 2021. Bacterial communities and functional genes stimulated during phenanthrene degradation in soil by bio-microcapsules. Ecotoxicol. Environ. Safety 212, 111970. https://doi.org/10.1016/j.ecoenv.2021.111970

Pápai M, Benedek T, Táncsics A, Bornemann TLV, Plewka J, Probst AJ, Hussein D, Maróti G, Menashe O, Kriszt B. Selective enrichment, identification, and isolation of diclofenac, ibuprofen, and carbamazepine degrading bacteria from a groundwater biofilm. Environ Sci Pollut Res Int. 2023, 30(15):44518-44535. doi: 10.1007/s11356-022-24975-6. Epub 2023 Jan 24.

Schaerer LG, Aloba S, Wood E, Olson AM, Valencia IB, et al. (2024) Enriched microbial consortia from natural environments reveal core groups of microbial taxa able to degrade terephthalate and terphthalamide
. PLOS ONE 19(12): e0315432. https://doi.org/10.1371/journal.pone.0315432

1. Supplementary chemical-structure table (Table S3) appears internally inconsistent and likely contains errors.
   Several entries (formulas/weights/structures) appear chemically inconsistent with diclofenac transformation chemistry. This undermines confidence in TP nomenclature used in the main text. Table S3 must be carefully verified and corrected.

Table S3 has been revised and modified.

1. Metabolite evidence and quantification for TP339 (NO₂-DCF) are insufficient to support strong toxicity attribution.
   TP339 is tracked via MRM conditions referenced to prior literature, without reporting confirmation by standards/HRMS, and results appear semi-quantitative (peak areas). Yet the discussion attributes increased toxicity to specific metabolites. This needs clearer qualification, improved confirmation, and/or more cautious conclusions.

We appreciate the reviewer’s comment. Indeed, TP339 (NO₂-DCF) has not been confirmed using an analytical standard, as it is not commercially available, and therefore its quantification was performed semi-quantitatively, following the MRM conditions reported in the literature. These analyses were performed by the research group led by Dr. Esteban Alonso (ORCID: 0000-0002-1647-9226), who has extensive experience in the field of Industrial and Environmental Chemical Analysis. As mentioned in the manuscript, information on the toxicity of metabolites generated during diclofenac biodegradation, particularly nitrogen-containing products, is limited.

For this reason, we have explicitly reflected this uncertainty in the conclusions:

“The observed toxicity was attributed to both identified (4´-OH-DCF and NO₂-DCF) and also possible unidentified metabolites, emphasizing the importance of evaluating metabolite formation and toxicity.”

In this way, the toxicity is not attributed solely to TP339, but it is acknowledged that both identified and potentially unidentified metabolites may contribute to the observed effect. We believe that this wording adequately reflects the analytical limitations and avoids overly strong conclusions.

1. Biosafety and feasibility are not adequately addressed.
   One isolate is reported as P. aeruginosa (opportunistic pathogen). Any implication of practical bioaugmentation should include biosafety/regulatory considerations, or the strain should be repositioned as a model organism for pathway discovery rather than a candidate for application.

We acknowledge that Pseudomonas aeruginosa is an opportunistic pathogen; although the strain isolated from the environment is not expected to be cytotoxic or pathogenic under normal conditions, its direct use in bioaugmentation would require strict biosafety measures. In this study, the strain isolated from an environmental matrix was used in laboratory experiments to help predict what could occur under real conditions. P. aeruginosa is commonly found in the environment, and studies like this demonstrate that this strain is capable of carrying out DCF biodegradation. We have clarified this in the manuscript to avoid suggesting that it could be applied directly in environmental settings, and the findings may guide the future selection of safe strains for bioaugmentation.

This sentence “Although P. aeruginosa was isolated from an environmental sample and is not expected to be cytotoxic or pathogenic under normal conditions, it is an opportunistic pathogen; therefore, its use in environmental bioaugmentation would require strict biosafety measures. In this study, it was employed as a laboratory model to investigate DCF biodegradation capacity” has been included in Lines 497-501.

1. Environmental relevance of the experimental conditions needs stronger framing.
   The use of 10 mg L⁻¹ DCF and glucose at 3 g L⁻¹ reflects an enrichment/high-load scenario rather than typical environmental conditions. The manuscript should explicitly state this limitation and avoid overgeneralizing to real wastewater or surface-water concentrations without supporting tests.

10 mg/L is indeed higher than typical environmental concentrations. However, we have demonstrated that the bacterium Pseudomonas sp. (P. syringae in the previous version of the manuscript) and the microbial consortium used in this study were able to completely degrade even high DCF concentrations in solution. Moreover, several previous studies have employed similar or even higher diclofenac concentrations. For example, Poddar et al. (2024) used 500 mg/L; Ivshina et al. (2019) used 50 mg/L; Stylianou et al. (2018) used 70 mg/L—all higher than the concentrations used in our work. Additionally, Bessa et al. (2017) employed the same concentration as our study, 10 mg/L. Using elevated concentrations in laboratory studies is a common practice to ensure measurable biodegradation and to assess the full capabilities of microorganisms under controlled conditions. We also note that the addition of glucose at 3 g L⁻¹ was intended to serve as a co-substrate; although this concentration is higher than would typically be found in the environment, it could be safely applied in a real treatment scenario if needed.

However, we acknowledge that this should be clearly indicated in the manuscript. Therefore, the following sentence has been included:

“The concentration used for the biodegradation studies in the present work is higher than those found in WWTPs and other environmental matrices, to ensure measurable biodegradation and assess the capabilities of microorganisms under controlled conditions. Other authors followed similar approaches (Bessa et al., 2017; Stylianou et al., 2018; Ivshina et al., 2019; Poddar et al., 2024).” Lines 615-619.

References

Stylianou, K.; Hapeshi, E.; Vasquez, M.I.; Fatta-Kassinos, D.; Vyrides, I. Diclofenac Biodegradation by Newly Isolated Klebsiella Sp. KSC: Microbial Intermediates and Ecotoxicological Assessment. J. Environ. Chem. Eng. 2018, 6, 3242–3248. https://doi.org/10.1016/j.jece.2018.04.052

Ivshina, I.B.; Tyumina, E.A.; Kuzmina, M.V.; Vikhareva, E.V. Features of diclofenac biodegradation by Rhodococcus ruber IEGM 346. Sci. Rep. 2019, 9, 1–13. https://doi.org/10.1038/s41598-019-45732-9.

Poddar, K.; Sarkar, D.; Bhoi, R.; Sarkar, A. Biotransformation of diclofenac by isolated super-degrader Pseudomonas sp. DCα4: Postulated pathways, and attenuated ecotoxicological effects. Environ. Pollut. 2024, 344, 123388. https://doi.org/10.1016/j.envpol.2024.123388.

Bessa, V.S.; Moreira, I.S.; Tiritan, M.E.; Castro, P.M.L. Enrichment of bacterial strains for the biodegradation of diclofenac and carbamazepine from activated sludge. Int. Biodeterior. Biodegrad. 2017, 120, 135–142. https://doi.org/10.1016/j. ibiod.2017.02.008.

Specific comments 

Major revisions

1. Replication and interpretation of metabarcoding
• Line 753–758: Only two MC samples are compared (“beginning and end”). Please either (i) add biological replicates for sequencing (recommended), or (ii) explicitly label the sequencing results as descriptive/hypothesis-generating and remove/soften causal language (e.g., “selection”, “positive selection”, “using DCF as carbon source”).

We assume that the mentioned lines (“753–758”) are not related to this comment on DNA metabarcoding. As stated in a previous point, since we now do not have the possibility to add biological replicates to this study, we have decided to address the option ii toning down the related statements throughout the manuscript. See Lines 31, 918, 943-945, 976-977, 1031-1032, 1067, 1072-1074, 1102-1103.

• Line 800–806: “Positive selection” wording is too strong without replication. Rephrase to “increased in relative abundance during the incubation”.

This change has been made (Line 944).

• Line 324–331: Rarefaction depths differ strongly between bacterial and fungal datasets. Please discuss potential bias from low fungal reads and provide read statistics/rarefaction curves (main or supplement).

To address this comment, we have added a new supplementary table (Table S4) showing metadata of sequenced samples (description and accession numbers in the ENA project PRJEB98466), number of reads (after filtering the sequences assigned to the division Bacteria or the kingdom Fungi), as well as the alpha diversity data (ASV richness, Shannon, Inverse Simpson and Evenness). Table S4 supports different sections of the manuscript, including 2.2.7. and 3.5.

The low number of fungal ITS2 sequences achieved for the sample collected before the biodegradation experiment likely reflected the low fungal biomass present in this sample. This statement has been included in Lines 389-391.

1. Taxonomic identification of isolates
• Line 179–188: Identification relies on 16S rRNA BLASTn. For Pseudomonas, 16S often lacks species-level resolution. Please provide accession numbers and either support species-level identification with additional markers (e.g., gyrB, rpoD)/genome-based methods, or revise to Pseudomonas sp. where not robust.

Accession numbers are provided in Table 1. We have decided to revise Pseudomonas syringae to Pseudomonas sp., as although the BLASTn analysis of the 16S rRNA gene showed a query coverage of 100% and a 99.68% sequence identity with P. syringae, there are closest related GenBank sequences identified as Pseudomonas sp. that exhibit even higher percentage identity (see Table below). Therefore, species-level assignment cannot be considered robust based solely on 16S rRNA data. Consequently, we have updated all corresponding information throughout the manuscript and contacted NCBI to correct the sequence submission. This was a misidentification originated from the external company that performed the 16S rRNA gene sequencing. However, in the case of Pseudomonas aeruginosa, the percentage of identity was 100%; therefore, we decided to retain the species-level identification.

Information about this statement has been included in Lines 503-506.

Table

1. Biosafety and application claims
• Line 15–19 (Abstract) and Conclusions (Line 911–917): Given the reported P. aeruginosa isolate, explicitly discuss biosafety limitations for application and/or reframe the isolate as a laboratory model. Avoid language implying direct deployment without addressing risks.

This sentence has been included in the abstract section: “…employed as laboratory models to investigate DCF biodegradation capacity under a biosafety-aware framework.” (Lines 17-19).

This sentence has been included in the conclusions section: “…; however, as it is an opportunistic pathogen, its use is restricted to laboratory studies and requires biosafety-aware considerations, precluding its direct environmental application.” (Lines 1078-1080).

1. Metabolite identification/quantification and toxicity attribution
• Line 254–260: Clearly indicate which analytes were quantified with authentic standards versus screened without standards (semi-quantitative, peak area only).

We thank the reviewer for this comment. These analyses were performed by the research group led by Dr. Esteban Alonso (ORCID: 0000-0002-1647-9226), who has extensive experience in the field of Industrial and Environmental Chemical Analysis. Quantification using authentic analytical standards was performed only for DCF and for the metabolites 4´-OH-DCF and 5-OH-DCF. In contrast, additional metabolites/transformation products for which authentic standards were not available, namely 1-O-acylglucuronide diclofenac, 4,5-dihydroxydiclofenac, TP339 (nitration), TP323 (nitrosation), and 5-hydroxydiclofenac lactam, were screened following the methodology described by Osorio et al. (2014). These compounds were identified based on MRM transitions and retention times reported in the literature and are therefore reported in a semi-quantitative manner (peak area only). We have clarified this distinction in the revised manuscript to avoid ambiguity (“…and results for these compounds are reported in a semi-quantitative manner using peak area only” Line 338-339).

References

Osorio, V.; Imbert-Bouchard, M.; Zonja, B.; Abad, J.L.; Pérez, S.; Barceló, D.  Simultaneous determination of diclofenac, its human metabolites and microbial nitration/nitrosation transformation products in wastewaters by liquid chromatography/quadrupole-linear ion trap mass spectrometry. J.  Chromatograph.  A 2014, 1347, 63-71. http://dx.doi.org/10.1016/j.chroma.2014.04.058

• Line 725–729 & 731–744: Temper claims linking toxicity to NO₂-DCF and hydroxylated metabolites. Add explicit uncertainty statements (e.g., “tentative identification”, “relative signal only”, “no absolute concentration”).

In the case of the hydroxylated metabolites, concentrations were quantified using their authentic analytical standards, so it is appropriate to refer to absolute concentrations. However, in response to the reviewer’s suggestion, we have moderated statements linking toxicity to NO₂-DCF and hydroxylated metabolites, and we have added explicit uncertainty statements in the section “3.4. Ecotoxicity studies in diclofenac-contaminated aqueous samples” (Lines 825, 871, 885, 888), as recommended.

• Line 737–744: There appears to be a strain-label inconsistency: TU = 10.5 is attributed differently across text and conclusions. Please correct and ensure consistency throughout.

Thank you for the reviewer’s comment. You are correct that there was an error in the strain nomenclature: CSWD.1 was indicated when it should have been CSWD.2. This mistake has now been corrected.

“Based on the previously reviewed literature, it can be concluded that in the case of CSWD.2, the bacterial strain that achieved the lowest TU values (10.5), the observed toxicity could be attributed to the tentative presence of the metabolites 4´-OH-DCF and NO₂-DCF, which were detected at lower signals than in CSWD.1 and MC. The MC treatment showed an intermediate TU value (14.3), likely because the concentrations of 4´-OH-DCF and the peak area of NO₂-DCF were higher than those obtained with CSWD.2 inoculation. Finally, the application of CSWD.1 resulted in the highest TU value (20.9), not only due to the presence of 4´-OH-DCF and NO₂-DCF but also because of the remaining DCF (55% of the initial concentration), which persisted in solution at 5.5 mg L⁻¹.” (Lines 883-891).

In the case of the conclusions, the strain assignments were already correct.

Supplementary Table S3 accuracy

• Table S3: Verify and correct all formulas, molecular weights, and structures; correct terminology (see “nitrification” note below). Ensure TP naming is consistent between main text, figures, and supplementary material.

Table S3 has been revised and corrected.

1. Environmental relevance / framing
• Line 498–500 and Methods Line 205–206: Add a short justification/limitations statement about using glucose (3 g L⁻¹) and 10 mg L⁻¹ DCF, clearly positioning the study as enrichment/high-load and not directly representative of typical environmental conditions.

This information has been included in Lines 615-619.

“The concentration used for the biodegradation studies in the present work is higher than those found in WWTPs and other environmental matrices, to ensure measurable biodegradation and assess the capabilities of microorganisms under controlled conditions. Other authors followed similar approaches (Bessa et al., 2017; Stylianou et al., 2018; Ivshina et al., 2019; Poddar et al., 2024).”

Minor revisions 

1. Template text left in manuscript
• Line 907–909: Delete the sentence “This section may be divided by subheadings…”, which appears to be leftover template guidance.

The correction has been made.

1. Figure caption inconsistency
• Line 843–846 (Figure 6 caption): Caption text appears to refer to bacterial genera when the figure is discussed as fungal. Please correct caption wording to match the actual figure content.

The correction has been made (Lines 999-1003).

1. Terminology
• Table S3: Replace “nitrification” with “nitration”/“nitrosation” (nitrification is a microbial nitrogen-cycle process, not a chemical substitution on the DCF ring).

The correction has been made in Table S3.

1. Microtox method clarity

• Line 262–273: Please clarify controls for matrix effects (pH, salinity, MSM components, glucose) and confirm that abiotic controls underwent the same incubation. Reporting pH at sampling (or stating adjustment) would strengthen interpretability.

The abiotic control was conducted under the same conditions as the treatments, but without bacterial inoculum. That is, the abiotic control consisted of MSM containing 10 mg L⁻¹ DCF and 3 g L⁻¹ glucose, with a pH of 7.0 ± 0.2.

Text in the manuscript has been modified: “For that purpose, supernatant samples collected at the beginning (abiotic control, consisting of MSM spiked with 10 mg L⁻¹ DCF and 3 g L^-1 of glucose) and at the end of the 28-day biodegradation experiment (treatments with P. aeruginosa CSWD.1, Pseudomonas sp. CSWD.2, and the microbial consortium MC). All samples exhibited an initial pH of 7.0 ± 0.2, which was not modified during incubation” Lines 344-349.

1. Data availability statement

Ensure the sequencing accession information is complete and that raw reads plus sample metadata are deposited. Confirm that sample IDs match those used in supplementary ASV tables/legends.

We confirm that all relevant information for DNA metabarcoding (sample metadata, raw sequences, etc.) is complete and well presented in the manuscript. We have added a new supplementary table (Table S4) including metadata of sequenced samples (description and accession numbers in the ENA project PRJEB98466).

We have added the following sentence in the section “Data availability statement”: “Raw sequencing data are available at the European Nucleotide Archive (ENA), EMBL-EBI, under the study accession no. PRJEB98466 (https://www.ebi.ac.uk/ena/browser/view/PRJEB98466).”

To clarify: The two supplementary lists of bacterial and fungal ASVs (Table S5 and S6) do not include sample IDs, as they are organized by ASV IDs instead.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript reports on Diclofenac biodegradation by bacterial strains and microbial consortium from activated sludge. This is written well with multi-aspect study of the case that makes it interesting. Some detailed comments to be addressed:

1)L19: Please move (45.5%) after removal.

2)L20: Was this intracellular or extracellular? Not indicated.

3)L25-26: This is not clear to the audience: ‘…to combine with the strains described here.’

4)L58-65: Can authors include chemical relevant formula/structure to be more understandable to the readers who are interested in chemistry aspect of the molecules?

5)Novelty of the study should be highlighted at the end of Introduction section.

6)The vessel used for biodegradation should be explained in more detail. Was  it baffled? Any aeration? How it was covered? Rubber seal or cotton?

7)Figure 1: Error bars need to be indicated under title what they stand for? SD or SE?

8)Figure 3: Can authors include error bars for the mean values on the graphs?

9)Table 3: R square values should have up to 3 digits in this table and all the text.

10)Table 3: In the caption, k1 , k2 and DT50 need to be described.

Author Response

REVIEWER 3

The manuscript reports on Diclofenac biodegradation by bacterial strains and microbial consortium from activated sludge. This is written well with multi-aspect study of the case that makes it interesting. Some detailed comments to be addressed:

1)L19: Please move (45.5%) after removal.

The correction has been made (Line 20)

2)L20: Was this intracellular or extracellular? Not indicated.

The metabolites were extracellular since they were identified in the supernatant. This has been clarified on Lines 21-22.

3)L25-26: This is not clear to the audience: ‘…to combine with the strains described here.’

This sentence has been modified to …” to combine with the microorganisms isolated in this study” (Lines 27-28).

4)L58-65: Can authors include chemical relevant formula/structure to be more understandable to the readers who are interested in chemistry aspect of the molecules?

Chemical formula/structure of DCF and its metabolites/transformation products have been included in Table S3.

5)Novelty of the study should be highlighted at the end of Introduction section.

This paragraph has been included at the end of Introduction section:

“This study offers interdisciplinary insights by integrating microbiology, analytical chemistry, toxicology, and metabarcoding to advance the understanding of DCF bio-degradation, a contaminant that remains insufficiently characterized in terms of its microbial degraders, transformation pathways, and associated toxicity.” (Lines 158-161).

6)The vessel used for biodegradation should be explained in more detail. Was  it baffled? Any aeration? How it was covered? Rubber seal or cotton?

The biodegradation assay was carried out in 250 mL Erlenmeyer flasks, filled to only 25% of their capacity (50 mL), and incubated under orbital shaking at 180 rpm to ensure adequate mixing and oxygen transfer. The flasks were not baffled, and aeration was achieved because they were covered only with a sterile cotton plug. We have added this information regarding the cotton plug in the revised Materials and Methods section (Lines 256-257).

7)Figure 1: Error bars need to be indicated under title what they stand for? SD or SE?

This sentence has been included in the figure legend of Figure 1: “Error bars indicate standard deviation from the replicates.”

8)Figure 3: Can authors include error bars for the mean values on the graphs?

The error bars are included in the figure; however, the errors are very small, and in some cases, the marker is even greater than the error bar. Upon close inspection, some error bars are visible, particularly in the case of Pseudomonas sp. CSWD.2.

9)Table 3: R square values should have up to 3 digits in this table and all the text.

The R-squared values have been modified up to 3 digits.

10)Table 3: In the caption, k1 , k2 and DT50 need to be described.

K1 and k2 have been described as *Degradation rate constants. However, DT50 was already described as **Time to decline to half the initial concentration of DCF.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

Thank you very much for considering to improve the manuscript. Reading carefully the new version of "Enhanced Diclofenac Biodegradation by Bacterial Strain and Microbial Consortium from Activated Sludge: Toxicity Assessment and Insights into Microbial Community Dynamics" I have noticed few things that in my opinion should be considered. Please find them listed below:

• L57-63: I think that the manuscript could use these to better explain the  recalcitrance mechanisms
• L91-99: Please consider to better discuss the relevance of these laboratory experiments with such high concentrations 
• L94-95: Please be more specific, which N-C bond? What specific hydrolase families the manuscript refer? It refer to amidases, aminohydrolases, or other specific enzyme classes? Please consider to specific
• In my opinion the objectives should be more specific and more accurate in formulation, For example, at O1 the manuscript should state the slection criteria considered. Further which pathways are targeted for elucidation? Which specific organisms or endpoints are considered? When presenting the objectives please avoid vague generic statement. They should be clearly formulated and presented 
• L158-161: I think that here is necessary also to briefly explain how mentioned disciplines will be integrated
• Please consider that the Statistical analysis subsection should be a stand alone subsection that cover all involved/considered statistical methods/approaches used for the obtained experimental data relevance and reliability tests
• L199: Please justify this "3 g L⁻¹ of additional carbon sources (glucose or yeast extract)". Please consider that high glucose/yeast extract concentrations may enable growth of non-degrading organisms; induce catabolite repression of DCF-degrading enzymes; and/or mask true DCF degradation capability
• L186-203: Please better evidence the biological replicates for enrichment cultures. Further, I understand based on those declared in the manuscript: degrading MC and 4 isolates were obtained - but this suggests for me a single enrichment culture (?) Please consider to clarify
• L205-207: Please provide justification for the 8.3-fold concentration increase on agar plates. Please consider that 200 mg/L in my opinion is extremely high and its effect may be inhibitory rather than selective
• L204-213: how many rounds were considered for successive colony isolation? how many different morphotypes? based on what criteria were considered? Further is very important to better describe of how microbial consortium was maintained
• In my opinion statistical evaluation of presented results are needed !!
• L417-418: In my opinion without statistical validation, the reliability of the 46% degradation value cannot be assessed. This is against to the basic principles of any quantitative analysis
• L420-421: Without support data this is poorly speculative
• Please consider that for any mechanistic claims these must to be sustained by experimental data and validation. Citing literature not enough. Please consider that through the whole manuscript where the case is!
• Also the manuscript must consider that tolerance is not equivalent with degradation capability. A strain may tolerate high DCF concentrations without degrading it, or vice versa. Please avoid to mix these concepts
• Based on those stated before I recommend a better consideration for the conclusion section

Comments on the Quality of English Language

improvements is needed !

Author Response

Reviewer 1

Dear Authors,

Thank you very much for considering to improve the manuscript. Reading carefully the new version of "Enhanced Diclofenac Biodegradation by Bacterial Strain and Microbial Consortium from Activated Sludge: Toxicity Assessment and Insights into Microbial Community Dynamics" I have noticed few things that in my opinion should be considered. Please find them listed below:

• L57-63: I think that the manuscript could use these to better explain the  recalcitrance mechanisms

We thank the reviewer for this comment. Numerous studies have reported the low removal efficiency of diclofenac (DCF) in conventional wastewater treatment plants (WWTPs); however, many of them do not provide a detailed discussion of the underlying mechanisms responsible for this recalcitrance. To address this point, we have expanded the discussion in lines 58–85 by incorporating additional mechanistic explanations and relevant literature that support the persistence and recalcitrant behavior of DCF in biological treatment systems

• L91-99: Please consider to better discuss the relevance of these laboratory experiments with such high concentrations 

The high concentration was intentionally used for laboratory-level studies to efficiently evaluate the capabilities of the isolated strain under controlled conditions. It is worth noting that basic research involves working across a wide range of concentrations, whereas environmental applications occur at much lower levels. Future experiments with real wastewater will be conducted at concentrations on the order of nanograms and micrograms per liter, which are more representative of environmental conditions.

• L94-95: Please be more specific, which N-C bond? What specific hydrolase families the manuscript refer? It refer to amidases, aminohydrolases, or other specific enzyme classes? Please consider to specific

We thank the reviewer for this insightful comment. In the current revised manuscript, we have clarified that the genomic analyses of Pseudomonas sp. DCα4 identified several genes encoding amidohydrolases (Lines 107-116).

• In my opinion the objectives should be more specific and more accurate in formulation, For example, at O1 the manuscript should state the slection criteria considered. Further which pathways are targeted for elucidation? Which specific organisms or endpoints are considered? When presenting the objectives please avoid vague generic statement. They should be clearly formulated and presented 

We thank the reviewer for this comment. The objectives of the study have been revised to be more specific and accurately formulated (Lines 169-182).

• L158-161: I think that here is necessary also to briefly explain how mentioned disciplines will be integrated

We thank the reviewer for this comment. We can clarify here how the different disciplines are integrated: microbiology provides enrichment cultures and microbial isolates, analytical chemistry enables the quantification of DCF and identification of metabolites, ecotoxicology evaluates changes in toxicity before and after biodegradation, and metabarcoding links microbial community composition to observed degradation patterns. Together, these approaches offer a comprehensive understanding of DCF biodegradation by connecting microbial actors, transformation pathways, and ecotoxicological outcomes. Considering that the study objectives have been revised and are now clearly formulated, we believe that this paragraph already provides sufficient context and that no additional detail is required in the manuscript.

• Please consider that the Statistical analysis subsection should be a stand alone subsection that cover all involved/considered statistical methods/approaches used for the obtained experimental data relevance and reliability tests

In the original version of the manuscript, the “Statistical analysis” subsection was included within Materials and Methods. However, following the reviewers’ comments, we decided to remove it and instead describe the statistical procedures directly within each methodological subsection where they were applied.

Below we attach the comment included in the response to reviewers:

“Statistical analysis – this subsection has been removed. For greater clarity, the statistical procedures are now described directly within each methodological subsection where they were applied.”

Nevertheless, a separate section dedicated to statistical analysis has been included (Lines 413-432).

L199: Please justify this "3 g L⁻¹ of additional carbon sources (glucose or yeast extract)". Please consider that high glucose/yeast extract concentrations may enable growth of non-degrading organisms; induce catabolite repression of DCF-degrading enzymes; and/or mask true DCF degradation capability

We also note that the addition of glucose at 3 g L⁻¹ was intended to serve as a co-substrate. Although this concentration is higher than would typically be found in environmental matrices, it could be safely applied in real treatment scenarios if required. Moreover, several studies cited in the manuscript have employed similarly high glucose concentrations as a cometabolite, for example, 1.5 g L⁻¹ (Żur et al., 2021) or 3 g L⁻¹ (Pápai et al., 2023, 2024). This clarification has been included in Lines 226–228.

References

Żur, J.; Marchlewicz, A.; Piński, A.; Guzik, U.; Wojcieszyńska, D. Degradation of diclofenac by new bacterial strains and its influence on the physiological status of cells. J. Hazard. Mater. 2021, 5, 403:124000. https://doi.org/10.1016/j.jhazmat.2020.124000

Pápai, M.; Benedek, T.; Sörös, C.; Háhn, J.; Csenki, Z.; Bock, I.; Táncsics, A.; Kriszt, B. Biotransformation of diclofenac by Stenotrophomonas humi strain DIC_5 and toxicological examination of the resulting metabolites. Appl. Microbiol. Biotechnol. 2024, 11;108(1):485. https://doi.org/10.1007/s00253-024-13320-1.

Pápai, M.; Benedek, T.; Táncsics, A.; Bornemann, TLV.; Plewka, J.; Probst, A.J.M.; Hussein, D.; Maróti, G.; Menashe, O.; Kriszt, B. Selective enrichment, identification, and isolation of diclofenac, ibuprofen, and carbamazepine degrading bacteria from a groundwater biofilm. Environ. Sci. Pollut. Res. Int. 2023, 30(15):44518-44535. https://doi.org/10.1007/s11356-022-24975-6.

• L186-203: Please better evidence the biological replicates for enrichment cultures. Further, I understand based on those declared in the manuscript: degrading MC and 4 isolates were obtained - but this suggests for me a single enrichment culture (?) Please consider to clarify

This statement has been clarified in the manuscript (Lines 221-223).

• L205-207: Please provide justification for the 8.3-fold concentration increase on agar plates. Please consider that 200 mg/L in my opinion is extremely high and its effect may be inhibitory rather than selective

The 8.3-fold increase in DCF concentration on agar plates (200 mg L⁻¹) was used to apply strong selective pressure to enrich for microorganisms capable of tolerating and potentially degrading DCF. We note that compound bioavailability on solid agar is lower than in liquid cultures, so the nominal concentration on plates does not directly translate to inhibitory conditions in solution. Although no specific studies exist for DCF, the diffusion and availability of compounds (e.g., antibiotics or biocides) in agar can be limited by their penetration and diffusion within the gel, affecting how cells experience these compounds compared to liquid media (O’Reilly et al., 2025). Moreover, the 200 mg L⁻¹ concentration corresponds to the same concentration used in the enrichment cultures conducted in parallel, ensuring consistency between solid and liquid selection conditions.

Reference

O’Reilly, P., Loiselle, G., Darragh, R. et al. Reviewing the complexities of bacterial biocide susceptibility and in vitro biocide adaptation methodologies. npj Antimicrob Resist 3, 39 (2025). https://doi.org/10.1038/s44259-025-00108-0

• L204-213: how many rounds were considered for successive colony isolation? how many different morphotypes? based on what criteria were considered? Further is very important to better describe of how microbial consortium was maintained

It is difficult to provide a precise answer to the reviewer’s questions, as the number of rounds required to obtain pure cultures depended on several factors, including the type of microorganism, its spatial distribution on the plate, the number of different microbial species present, etc.

Similarly, 18 different isolates were found in total. For this reason, in the Materials and Methods section, we specified the criteria considered for selection (colony size, color, edge, and elevation), but this high number of isolates does not make it feasible to describe these parameters for each colony.

Finally, the microbial consortium was maintained in the same way as the isolates, as detailed in Lines 239–240.

• In my opinion statistical evaluation of presented results are needed !!

A statistical analysis has been performed for all experiments conducted in this study.

• L417-418: In my opinion without statistical validation, the reliability of the 46% degradation value cannot be assessed. This is against to the basic principles of any quantitative analysis

Experiments were performed in triplicate, and the standard deviation is included in Figure 1. However, we have decided to modify the text to read “…nearly half of the initial content was removed” (Line 451).

• L420-421: Without support data this is poorly speculative

The reviewer is true, but we don’t have any other way to check the plateau in DCF degradation.

• Please consider that for any mechanistic claims these must to be sustained by experimental data and validation. Citing literature not enough. Please consider that through the whole manuscript where the case is!

We would like to clarify that the experiments conducted in this study were focused on the objectives defined, which included enrichment and isolation of DCF-degrading microorganisms, identification of metabolites, and evaluation of ecotoxicity. However, other mechanistic studies, such as investigating the specific reasons why biodegradation may halt, were beyond the scope of this work. In fact, monitoring DCF concentration during the enrichment experiments was performed solely to determine the optimal timing for isolating microorganisms. Anyway, we will take into account all these aspects for future work.

• Also the manuscript must consider that tolerance is not equivalent with degradation capability. A strain may tolerate high DCF concentrations without degrading it, or vice versa. Please avoid to mix these concepts

The reviewer is correct, and we have revised the manuscript to ensure that these concepts are not conflated.

• Based on those stated before I recommend a better consideration for the conclusion section

In the conclusions, only the MC consortium and the CSWD.1 and CSWD.2 strains are mentioned, all of which are capable of transforming DCF through the biodegradation studies. Therefore, we consider that there is no issue of conflating concepts in this section.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Thank you for the revised version and detailed responses. The manuscript has improved substantially, and the main concerns (taxonomic assignment, biosafety framing, experimental context, and several internal inconsistencies) have been addressed satisfactorily.

Before I can recommend acceptance, please implement the following minor, but important textual/formatting adjustments to ensure consistency and avoid over-interpretation:

1. TP339 / NO₂-DCF wording
   Please revise any remaining assertive statements (e.g., “confirming/corresponds to NO₂-DCF”) to clearly indicate a tentative/putative identification based on MRM/retention time comparisons from literature, given the lack of authentic standards (or HRMS confirmation). Keep conclusions about metabolite-specific toxicity appropriately cautious.

2. Data Availability Statement (line 1125-1126. pg 25)
   Please update the Data Availability Statement to explicitly include the ENA accession number (PRJEB98466) and clarify what has been deposited (raw reads and associated metadata), ensuring consistency with the Methods section.

3. Abstract – community interpretation
   Given the limited replication for metabarcoding, please avoid functional language such as “actively” and keep the statement descriptive (e.g., “may be associated with…” under the tested conditions).

Once these points are addressed, I would support acceptance.

Author Response

REVIEWER 2

Thank you for the revised version and detailed responses. The manuscript has improved substantially, and the main concerns (taxonomic assignment, biosafety framing, experimental context, and several internal inconsistencies) have been addressed satisfactorily.

Before I can recommend acceptance, please implement the following minor, but important textual/formatting adjustments to ensure consistency and avoid over-interpretation:

1. TP339 / NO₂-DCF wording
   Please revise any remaining assertive statements (e.g., “confirming/corresponds to NO₂-DCF”) to clearly indicate a tentative/putative identification based on MRM/retention time comparisons from literature, given the lack of authentic standards (or HRMS confirmation). Keep conclusions about metabolite-specific toxicity appropriately cautious.

All the aspects highlighted by the reviewer have been addressed:

Line 23: “…and putative NO₂-DCF…”

Line 24: “…and tentatively identified NO₂-DCF”

Lines 719–722: “…TP339 was tentatively identified as NO₂-DCF based on MRM transitions, with identification supported by characteristic fragment ions and retention time matching, consistent with previously reported data.”

Line 1124: “…putative NO₂-DCF…”

Line 1128: “…putative NO₂-DCF…”

1. Data Availability Statement (line 1125-1126. pg 25)
   Please update the Data Availability Statement to explicitly include the ENA accession number (PRJEB98466) and clarify what has been deposited (raw reads and associated metadata), ensuring consistency with the Methods section.

The Data Availability Statement has been updated.

1. Abstract – community interpretation
   Given the limited replication for metabarcoding, please avoid functional language such as “actively” and keep the statement descriptive (e.g., “may be associated with…” under the tested conditions).

This aspect has been modified (Line 31).

Once these points are addressed, I would support acceptance.

We would like to thank the reviewer for their comments, which have helped us improve the manuscript.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The comments and suggestions are addressed carefully. The manuscript is in good shape now and is suggested for publication.

Author Response

Reviewer 3

The comments and suggestions are addressed carefully. The manuscript is in good shape now and is suggested for publication.

We would like to thank the reviewer for their comments, which have helped us improve the manuscript.

Author Response File: Author Response.pdf